InteractiveFly: GeneBrief

senseless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The Lyra mutation was first described by Jerry Coyne in 1935. Lyra causes recessive pupal lethality and adult heterozygous Lyra mutants exhibit a dominant loss of the anterior and posterior wing margins. Unlike many mutations that cause loss of wing tissue (e.g., scalloped, Beadex, cut, and apterous), Lyra wing discs do not exhibit increased necrotic or apoptotic cell death, nor do they show altered BrdU incorporation. However, during wing disc eversion, loss of the anterior and posterior wing margins is apparent. senseless (sens), a gene that is necessary and sufficient for peripheral nervous system (PNS) development (Nolo, 2000), is allelic to Lyra. Lyra/Sens is a nuclear protein with four Zn fingers that is expressed and required in the sensory organ precursors (SOPs) for proper proneural gene expression. Ectopic expression of Lyra in many ectodermal cells causes induction of PNS external sensory organ formation and is able to recreate an ectopic proneural field. Hence, Lyra is both necessary and sufficient for PNS development. Proneural genes activate Lyra expression (Nolo, 2000 and 2001).

Lyra alleles are neomorphic alleles of sens that cause ectopic expression of Senseless in the wing pouch. Similarly, overexpression of Senseless in the wing disc causes loss of wing margin tissue, thereby mimicking the Lyra phenotype. Lyra mutants display aberrant expression of Delta, Vestigial, Wingless, and Cut. As in Lyra mutants, overexpression of Senseless in some areas of the wing pouch also leads to loss of Wingless and Cut. Thus overexpression of Senseless causes a severe reduction in Notch signaling that in turn may lead to decreased transcription of several key genes required for wing development, leading to a failure in cell proliferation and loss of wing margin tissue (Nolo, 2001).

The onset of PNS development is marked by the expression of proneural genes of the achaete-scute complex (AS-C), atonal, or amos in clusters of ectodermal cells, termed the proneural clusters. The SOP is then selected from a small group of cells (the proneural field) within the proneural cluster that accumulates higher levels of proneural proteins than neighboring cells. Eventually, the SOP accumulates the highest levels of proneural proteins. The proneural basic helix loop helix (bHLH) proteins (Scute, Achaete, Asense, Atonal, and Amos) then implement the neuronal differentiation pathway as functional heterodimers with the Daughterless protein (Nolo, 2000 and references therein).

The upregulation of proneural gene expression in the SOP and the downregulation in adjacent ectodermal cells is mediated through a signaling pathway in which the genes of the Notch pathway, including Notch (N), Delta (Dl), Suppressor of Hairless [Su(H)], and the Enhancer of split complex genes [E(spl)-C], play a pivotal role. Loss-of-function mutations in these genes cause most or all cells of the proneural cluster to assume the SOP fate. Although it has been proposed that accumulation of proneural gene products in the SOP via Notch signaling is stochastic, the SOP always occupies a very similar position in the cluster. It has therefore been proposed that prepatterning factors predetermine a small subset of cells in the proneural cluster. One of these cells will become the SOP by accumulating more proneural protein than its neighbors and will produce more Delta protein. Although the upregulation of proneural gene expression is thought to be a prerequisite for sensory organ development, it is thought that several other unidentified proteins are required for the process (Nolo, 2000 and references therein).

Lyra/Senseless is a novel proneural-like protein. Severe loss-of-function mutations in sens abolish the further upregulation and maintenance of proneural gene expression in the SOPs. Interestingly, ectopic expression of Sens induces ectopic PNS organs, causing large tufts of bristles. Sens is both necessary and sufficient for PNS organ development but seems to require the activity of proneural proteins. sens encodes a transcription factor that enhances and maintains proneural gene expression in the SOP and is able to create a proneural cluster when ectopically expressed. Sens is therefore an essential component of the proneural Notch signaling pathway required for proper SOP differentiation (Nolo, 2000).

The embryonic PNS of Drosophila consists of three types of sensory transducers: the multiple dendritic (md) neurons; the external sensory organs (es), and the chordotonal organs. The latter two types of organs consist of four cells: a neuron, a glial cell, and two support cells. These originate from the SOPI that produces the SOPII a and b. Mutations in sens cause an extensive loss of many cells of the embryonic PNS, as revealed by staining with anti-Couch Potato, a marker that labels all nuclei of the PNS cells. Labeling with monoclonal antibody 22C10, a neural-specific antibody, shows that most neurons are absent. The few neurons that remain are mostly of the md type, but the md neurons that are lost are dependent on scute, atonal, and amos. Staining with anti-Prospero, a marker for PNS glial cells, reveals a very severe loss of glial cells. Staining with anti-Cut shows a significant loss of es support cells. In summary, these data show that all types of PNS cells in sens mutants are affected and that most cells are absent in mature stage 16 embryos (Nolo, 2000).

In the embryonic PNS nervous system, Sens is expressed at low levels in some cells surrounding the SOPIs and at high levels in SOPIs, SOPIIs, and differentiating progeny. In imaginal discs, Sens expression is first observed at low levels in ectodermal cells in proximity to many SOPs. This domain may correspond to the proneural field, which has been shown to accumulate higher levels of Achaete-Scute expression than other cells of the proneural cluster. However, levels of Sens protein expression are dramatically enhanced in the SOPIs. Sens is expressed prior to a typical SOP marker, A101 lacZ, but enhanced expression of Sens in the presumptive SOP is often coincident with A101 lacZ expression (Nolo, 2000).

The SOPI and SOPII cells are present in sens mutant embryos (Salzberg, 1994). Hence, there are two possible alternatives to explain the phenotype: the SOPII cells fail to divide, or the SOPII cells and/or their progeny are eliminated by apoptosis. Double labeling with terminal deoxynucleotidyl transferase (TUNEL) and 22C10 shows dying cells in the PNS of stage 16 sens embryos but not in wild-type control embryos. Similar data were obtained by in situ hybridization with grim, a marker for apoptotic cells in Drosophila. Since no alterations are observed in the expression of dacapo, a cyclin-dependent kinase inhibitor expressed in terminally dividing cells of the PNS, it is concluded that the severe loss of PNS cells in late embryos is due to apoptosis of SOPIIs and/or differentiating cells of the PNS (Nolo, 2000).

If Senseless is required for proneural expression, ectopic expression of Sens may induce proneural gene expression. Indeed, ectopic expression of Sens using the dpp-GAL4 driver causes robust expression of Sens in the expected wing stripe. This expression causes the formation of numerous bristles and sensilla campaniforma in the adult wing in proximity of the third wing vein where dpp is normally expressed. Similarly, ectopic expression of Sens in the leg disc causes many supernumerary bristles in the sternopleural area as well as in more distal portions of the leg. Ectopic bristles are observed with all UAS-sens reporters. Some UAS-sens transgenes driven by dpp-GAL4 cause very severe tufting in the adult notum, wings, and legs, and loss of tissues in other portions of imaginal discs, e.g., wing margins and distal leg structures. It is concluded that ectopic expression of Sens is sufficient to initiate ectopic external sensory organ development (Nolo, 2000).

To determine the molecular cascade underlying the formation of the extra external sensory organs, wing discs of UAS-sens; dpp-GAL4 larvae were stained with anti-Scute antibodies. Ectopic Sens expression causes ectopic activation of Scute and Asense. Hence, Sens is able to activate proneural gene expression. This provides a molecular basis for the generation of additional external sensory organs, since ectopic proneural gene expression has previously been shown to be sufficient to induce ectopic PNS organ formation (Nolo, 2000).

If Sens induces proneural gene expression and proneural genes are required for Sens production, a super-additive or synergistic interaction between sens and proneural genes may occur. Therefore, the weakest UAS-sens transgene (C1) was expressed in combination with an UAS-scute and an UAS-atonal transgene under the control of dpp-Gal4. Overexpression of Scute or Atonal alone causes a relatively mild phenotype with relatively few extra bristles. Scute expression induces Sens expression, but the expression levels of Sens are lower than those induced by dpp-Gal4; UAS-sens. Ectopic expression of Sens with the dpp-Gal4 driver causes a stronger phenotype when compared to ectopic expression of Scute or Atonal. However, simultaneous overexpression of Sens and Scute or Atonal causes severe tufting, including in many areas where Scute, Atonal, or Sens, when expressed individually, does not normally cause ectopic bristles. These areas do correspond to areas where the dpp-Gal4 driver has previously been shown to be expressed. It is therefore concluded that sens and the proneural genes can interact in a synergistic fashion (Nolo, 2000).

The data so far imply that Sens is dependent on proneural gene expression and that proneural gene expression can be induced by Sens. This raises another issue: can overexpression of Sens in the absence of proneural genes produce external sensory organs? Since sens expression depends on the expression of many proneural genes and since Sens is able to induce ectopic expression of several proneural proteins, removal of one or several proneural genes may not be fully effective, i.e., all proneural genes should be removed to test this hypothesis. Flies that are mutant for achaete and scute (Df(1)sc10-1) are almost devoid of micro- and macro-chaetae. Overexpression of Sens (C8) in Df(1)sc10-1 flies causes few ectopic bristles when compared to a wild-type background, suggesting that Sens requires the presence of proneural genes (Nolo, 2000).

To determine whether the proneural genes require Sens, Scute was overexpressed in the dorsal portion of the wing disc by driving UAS-scute with apterous-GAL4 and mutant clones were induced that lack sens in these discs. Flies that are UAS-scute; apterous-GAL4 exhibit numerous thoracic extra bristles. Six flies were obtained that have large clones, and in each fly a complete loss of all micro- and macro-chaetae in the clone was observed. These data clearly suggest that Sens is required for bristle development, even when Scute is overexpressed (Nolo, 2000).

These data indicate that ectopic expression of Sens is a more potent inducer of supernumerary PNS organs than is ectopic expression of proneural genes using the same drivers. For example, ectopic expression of Sens at the anterior-posterior border (the dpp domain between future wing veins 3 and 4) causes numerous bristles and sensilla campaniforma along the length of the wing blade, except in the most proximal portion, the wing hinge region. In some areas, the width of the field contains as many as five adjacent PNS organs. Sens is able to induce Scute in the ventral and the dorsal area of the wing pouch as well as in the dorsal portion of the disc. However, the area of the wing hinge region is much less sensitive to overexpression of Sens. This suggests that Sens does not have the same inductive capacity in all cells and also further supports the idea that Sens requires proneural activity to induce PNS organs (Nolo, 2000).

Induction of Sens does not only induce Scute and Asense. Sens expression using the dpp-GAL4 driver alters Delta expression. The domain that normally gives rise to the third wing vein, is altered in Sens-overexpressing discs. Overexpression of Sens induces Delta expression ectopically in the dpp domain, broadening and intensifying the endogenous Delta domain. In addition, a consistent reduction of expression in the fourth wing vein domain is observed. This ectopic Delta expression is likely to be mediated by Scute/Asense overexpression (Nolo, 2000 and references therein).

To determine the relationship between Sens expression and the proteins of the Enhancer of Split complex, wild-type discs were stained for both proteins. There is little overlap between the two nuclear proteins. Cells that express Sens are intermingled with E(spl) expressing cells, but the majority of cells that express Sens do not express E(spl). Similar observations were also made with E(spl)m8-lacZ and with E(spl)m4-lacZ. These data indicate that Sens expression in cells fated to develop into SOPs is concomitant with the presence of E(spl) proteins, but that elevation of Sens expression and cell enlargement during SOP specification accompanies a rapid removal of the E(spl) protein. These data are also in agreement with the proposition that E(spl) is a negative regulator of proneural gene expression and that its downregulation permits SOP development (Nolo, 2000 and references therein).

Ectopic expression of Sens may not only activate the proneural genes and Delta but may recreate an ectopic proneural field. Expression of several E(spl) proteins depends on the presence of the proneural genes. Therefore Sens was overexpressed using the dpp-GAL4 driver in E(spl)m8-lacZ and E(spl)m4-lacZ imaginal discs. Wild-type discs contain proneural clusters that express cytoplasmic ßgalactosidase [E(spl)] in which few cells are Sens positive. Overexpression of Sens causes a strong induction of ßgalactosidase staining associated with E(spl)m4-lacZ and E(spl)m8-lacZ. This induction is not restricted to cells in which Sens is expressed but can be detected in adjacent cells as well. This indicates that Sens can induce in a cell-nonautonomous fashion E(spl) expression, probably by activating Delta expression. A more detailed cellular analysis shows that when Sens expression is elevated in a particular cell, ßgalactosidase levels are consistently low or absent. It is inferred that ectopic Sens leads to expression of the essential components required to establish a proneural domain in some areas of the wing discs. This ability is most likely mediated by its ability to activate the proneural genes. The wing hinge region is, however, refractory to induction of Scute, Delta, and E(spl) upon overexpression of Sens (Nolo, 2000).

Since ectopic Sens is able to induce E(spl) expression and since elevated Sens levels are associated with low levels or absence of E(spl) protein during SOP specification in normal and ectopic conditions, it was of interest to enquire how ectopic expression of both proteins in the same cells would affect PNS organ development. Since overexpression of E(spl) causes a loss of external sensory organs, the component that is most downstream in the pathway should be epistatic to the more upstream component. The dorsal portion of the thorax of a dpp-GAL4; UAS-sens fly has extra bristles. Scutellar bristles are lost in dpp-GAL4; UAS-E(spl)m8 flies. Coexpression of both Sens and E(spl)m8 proteins always leads to a very strong reduction in supernumerary bristles in most areas, occasionally loss of bristles. Hence, ectopic E(spl), counteracting neurogenesis, is epistatic to ectopic Sens, stimulating neurogenesis, in the pathway that specifies the SOP (Nolo, 2000).

Although loss of sens affects all cells of the embryonic PNS, it affects multiple dendritic neurons less than the external and chordotonal sensory organs. The loss of PNS cells in embryos seems to be at least in part mediated by cell death. In embryos, Sens is therefore required for terminal differentiation and/or viability of most or all PNS cells. Hence, the phenotype associated with loss of Sens is similar but less severe than that associated with loss of proneural genes. This may be due to another homolog of sens (CG11243: probability e-46) that is expressed in some cells of the PNS and CNS (Nolo, 2000).

As shown above, ectopic expression of Sens causes many ectodermal imaginal cells to take the SOP fate. This implies that Sens is sufficient to induce external sensory organ formation. The data also indicate that Sens is downstream of proneural gene activity, as their presence is required for its transcription. Sens protein is not required for proneural gene expression in proneural clusters, but its presence in the SOPs is necessary and sufficient to enhance and maintain proneural expression to specify proper neuronal fate of SOPs. This model is supported by numerous observations. (1) Sens expression is dependent on proneural gene expression; (2) ectopic expression of Sens induces proneural gene expression; (3) ectopic proneural gene expression induces Sens expression; (4) Sens and the proneural genes scute and atonal interact in a synergistic fashion; (5) loss of Sens leads to a failure to enhance and maintain proneural protein expression in the SOPs; (6) the ability of Sens to recruit ectodermal cells into the SOP pathway is severely impaired in achaete and scute mutants. These observations also provide a molecular basis for the observations that ectopic expression of different proneural genes, including Atonal, leads to the production of a variety of es organs. They may all activate Sens, which in turn activates different proneural genes when the cells are not in their normal cellular context. However, the data also suggest that Sens must play another role that is required for the development of adult sensory organs, since overexpression of Scute in the absence of Sens is unable to promote es organ formation. This may be due to the inability of the progeny of the SOPs to survive or the inability to repress E(spl) expression (Nolo, 2000).

The ability of ectopic Sens to induce external sensory organ formation is most likely due to its ability to cause expression of many key players that are normally expressed in the proneural cluster. Proneural genes activate the transcription of the E(spl) genes and sens. The Sens protein may then act via two pathways in the SOP. (1) It may directly activate proneural gene expression participating in the initiation and/or maintenance of an autoregulatory loop. This mode of action is supported by the observation that ectopic Sens can induce proneural gene expression in the absence of endogenous proneural proteins or E(spl) proteins. In addition, the proneural genes contain consensus binding sites for the Sens protein, suggesting that the interaction may be direct. (2) Sens may first enhance and subsequently inhibit transcription of E(spl) genes. Expression of the genes of the E(spl) complex is clearly reduced in the SOPs to permit their specification. It is proposed that Sens also plays a role in this process by inhibiting transcription of the E(spl) genes in the SOPs. This in turn may allow further upregulation of proneural gene expresssion, followed by ectopic expression of E(spl) in neighboring cells that do not express Sens, suggesting that they receive a signal from the proneural proteins expressing cells. This signal is most likely Delta, since Delta expression is clearly upregulated in the cells that express Sens. Cells that do not express or express very low levels of Sens then accumulate more E(spl) than those that do express higher levels of Sens. Hence, the reduction of Notch signaling in the SOP may be strongly enhanced by the presence of Sens to help specify SOPs. Indeed, ectopic coexpression of E(spl)m8 and Sens dramatically reduces the action of Sens and in some areas of the notum creates a phenotype that is typically associated with overexpression of E(spl)m8 alone, i.e., loss of bristles. This suggests that E(spl)m8 acts downstream of Sens. In ectodermal cells, Sens normally does not play a role because none of these cells acquire enough proneural gene expression to activate Sens at a level that is sufficient to activate proneural gene expression above a required threshold. The latter statement is supported by the expression pattern of Sens, which is restricted to those cells that express the highest levels of proneural proteins and by the observation that robust levels of ectopic proneural gene expression must be attained to induce ectopic Sens expression. In summary, it is proposed that the function of Sens is to integrate proneural gene expression into the Notch signaling pathway to promote proper SOP development in the Drosophila PNS (Nolo, 2000).

Ordered patterning of the sensory system is susceptible to stochastic features of gene expression

Sensory neuron numbers and positions are precisely organized to accurately map environmental signals in the brain. This precision emerges from biochemical processes within and between cells that are inherently stochastic. This study investigated impact of stochastic gene expression on pattern formation, focusing on senseless (sens), a key determinant of sensory fate in Drosophila. Perturbing microRNA regulation or genomic location of sens produced distinct noise signatures. Noise was greatly enhanced when both sens alleles were present in homologous loci such that each allele was regulated in trans by the other allele. This led to disordered patterning. In contrast, loss of microRNA repression of sens increased protein abundance but not sensory pattern disorder. This suggests that gene expression stochasticity is a critical feature that must be constrained during development to allow rapid yet accurate cell fate resolution (Giri, 2020).

An outstanding challenge is to determine whether stochasticity inherent to gene expression is transmitted across scales to vary the fidelity of pattern formation. This study has focused on the organization of sensory bristles along the adult wing margin. During pattern formation, cells experience noise in Sens protein copy number that derives from two sources. One source is from the discontinuous bursts of sens transcription, and the other source is from random birth-death events that affect sens mRNA and protein. For sens, as defined by the 19.2 kb region constituting the transgene, this intrinsic noise was not sufficient to transmit disorder to the adult pattern. However, when sens was subject to trans regulation between paired alleles, protein noise was greatly enhanced, which was sufficient to disorder the adult pattern. Therefore, trans interaction between paired homologs is an unanticipated source of noise. It is tempting to speculate that trans regulation might be a natural means to modulate gene expression noise (Giri, 2020).

Allelic pairing has been observed across multiple organisms. Pairing often precedes trans regulatory interactions, such as X-inactivation. In Drosophila, pairing and trans regulation between homologs has been demonstrated for several developmental genes. Indeed, trans allelic interactions appear to be a pervasive feature of the Drosophila genome. However, pairing of homologous alleles is not necessarily indicative of trans regulation (Giri, 2020).

It remains to be determined how pairing at 57F5 enhances Sens output and noise. The noise profile observed for paired sens alleles at 57F5 can be partly modeled as the effect of enhanced transcription burst size. Since the two-state model uses general rate parameters k_on, k_off, and S_m to regulate bursting, it is agnostic to the specific molecular processes that direct transcription. Distinct mechanisms such as chromatin remodeling, enhancer looping, transcription factor binding-unbinding, or preinitiation complex assembly-disassembly might be rate-limiting for k_on, k_off, or S_m at different levels of Sens expression. Thus, the complex noise profile observed for 57F5 might be a signature of multi-state transcription. Indeed, multi-state transcription kinetics have been observed for other genes. It is also possible that certain types of mRNA state transitions could generate an enhanced profile of protein noise. These might include transitions from an unspliced to spliced state, cytoplasmic mRNA processing, differential translation efficiencies, or toggling between reversible translating and non-translating states. Therefore, although the modeling performed in this study implicates transcriptional bursting as a major source of protein noise, it remains to be experimentally verified. Use of transgenes inserted into only two sites should be augmented with comparison to more sites and to the endogenous sens gene. Examining transcription bursting via smFISH or MS2-MCP tagged RNA at paired and unpaired loci will be necessary to determine if bursting kinetics are different for different genomic locations (Giri, 2020).

Noise levels differ between 22A3 and 57F5 cells only in the expression regime of 300-800 Sens molecules. Yet, this difference appears to affect pattern order-disorder. It would suggest that a subset of cells with fewer than 800 Sens molecules are at a developmental decision point between epidermal (E) and sensory (S) fates. This is an order of magnitude less than the approximately 8,000-10,000 Sens molecules observed in terminal S-fated cells. Stochastic fluctuations have the largest impact when protein copy numbers are low. Yet, production of a large number of proteins would raise the time and metabolic cost required to undergo developmental transitions. It suggests that expression noise of certain genes is optimized to allow accurate pattern formation without requiring the production of large numbers of fate-determining proteins (Giri, 2020).

How might this optimization be realized? Individually varying the different rate constants in a two-state model produces very different protein noise-output relationships. Regulating k_on provides the most effective way to increase protein output without increasing noise. Increasing k_on increases the frequency of transcriptional bursts without increasing burst size. Indeed, smFISH experiments show that while sfGFP-sens transcription burst size is constant across the wing margin, mRNA output is regulated by tuning k_on and burst frequency across cells. A similar regulatory mechanism is inferred for sens transcription by coupling protein noise measurements to stochastic models (Giri, 2020).

Proper inference of transcription kinetics using protein measurements requires a straightforward correlation between mRNA and protein numbers. Such correlations have been noted. For instance, bicoid mRNA and protein numbers are reproducible to within 10% and scale proportionately with gene dosage. Stochastic models of transcription and protein production were used to correctly infer mRNA and protein copy numbers for bacteriophage lambda repressor CI and the HIV-1 Long Terminal Repeat promoter. Indeed, protein reporters have been successfully used to infer transcriptional bursting parameters k_on and k_off for a wide variety of transgenic and endogenous mammalian genes (Giri, 2020).

The results suggest that pattern disorder is driven by Sens protein noise rather than protein levels. This might seem counterintuitive since there are many examples of gene overexpression causing developmental phenotypes. The explanation likely lies in the mechanism of wing margin patterning, which occurs in two stages. First, the Wg morphogen induces Sens expression leading to tens to hundreds of protein molecules per cell. Second, Sens expression is self-organized into a periodic row of S and E cells by Delta-Notch mediated lateral inhibition. If all cells should express Sens to an abnormally high level, lateral inhibition still acts on the relative differences between cells to properly resolve the pattern. Consistent with this, reducing endogenous sens gene dose to one copy does not affect bristle pattern formation. On the other hand, enhanced fluctuations in Sens output appear to cause pattern disorder. The simplest interpretation is that during the second stage, cell-to-cell transmission and reception of inhibitory signals is distorted by high intrinsic fluctuations in Sens. If fluctuations are large enough to trigger positive feedback between proneural factors, it would render a cell resistant to lateral inhibition. The net outcome would be cells that spontaneously adopt S fates out of order (Giri, 2020).

Stochastic transcriptional fluctuations have been harnessed in bet hedging systems such as induction of lactose metabolism in E. coli) and cell competence in B. subtilis. In mammalian cells, stochastic fluctuations confer phenotypes such as HIV latency periods and acquisition of cancer drug resistance. In developmental contexts, variability in transcription factor output has been shown to affect cell-fate switches that rely on absolute concentration thresholds. However, some developmental systems buffer against expression stochasticity by relying on relative changes in expression or intercellular signaling. This study shows that a patterning system relying on lateral inhibition can buffer against tissue-scale changes in protein output, but is sensitive to stochastic fluctuations in protein copy numbers (Giri, 2020).

REGULATION

Promoter

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Ordered patterning of the sensory system is susceptible to stochastic features of gene expression

Sensory neuron numbers and positions are precisely organized to accurately map environmental signals in the brain. This precision emerges from biochemical processes within and between cells that are inherently stochastic. This study investigated impact of stochastic gene expression on pattern formation, focusing on senseless (sens), a key determinant of sensory fate in Drosophila. Perturbing microRNA regulation or genomic location of sens produced distinct noise signatures. Noise was greatly enhanced when both sens alleles were present in homologous loci such that each allele was regulated in trans by the other allele. This led to disordered patterning. In contrast, loss of microRNA repression of sens increased protein abundance but not sensory pattern disorder. This suggests that gene expression stochasticity is a critical feature that must be constrained during development to allow rapid yet accurate cell fate resolution (Giri, 2020).

The irreversible progression from a disordered to an ordered arrangement of cells within tissues is a hallmark of development. Developing organisms rely on precise control of cellular gene expression in order to achieve this outcome. However, biochemical reactions such as transcription and translation involve stochastic molecular collisions subject to intrinsic variability. Therefore, a central question in developmental biology concerns how probabilistic gene expression generates deterministic developmental outcomes (Giri, 2020).

Fluctuations in mRNA and protein numbers occur because of random birth and death of these molecules. Since one molecule of mRNA is usually translated into multiple copies of proteins, small fluctuations in mRNA number can lead to larger fluctuations in protein number. In theory, the stochasticity in protein copy number caused by birth-death processes will be mitigated if large numbers of protein molecules are present in each cell. Indeed, many transcription factors are reported to be expressed in excess of 104-105 protein copies in terminally fated cells. However, it is unclear how many copies of such fate-determining proteins are present at cell-fate decision points, and therefore how extensively the stochasticity inherent to birth-death processes impinges upon fate decisions (Giri, 2020).

There are additional sources of noise in gene expression. Many genes are transcribed in bursts. Such genes switch stochastically between an actively transcribing state and an inactive non-transcribing state. This generates bursts of newly synthesized mRNA molecules interspersed with periods of dormancy. Various physical features of gene promoters, their enhancers, and the transcription factors that bind to them have been shown to affect the burstiness of gene transcription. Several mechanisms have been proposed to buffer protein numbers against bursty mRNA fluctuations. These include spatial and temporal averaging of transcript numbers, polymerase pausing, and autoregulation of gene expression (Giri, 2020).

Since tissues are patterned by the actions of gene regulatory networks (GRNs) across diverse temporal and spatial-scales, efforts are being made to understand how stochastic expression of these genes affects pattern formation. This study has focused on a patterning system involving the Wnt and Notch signaling pathways, which are two widespread means for cells to communicate with one another (Giri, 2020).

Often, Wnt and Notch signals intersect upon a set of cells, and from this emerge precise patterns of differentiated cells. A classic example of such an intersection is the emergence of rows of sensory organ (S) cells located alongside the dorsal and ventral (DV) compartment boundary of the Drosophila wing imaginal disc. Each row of S fated cells develops into a highly ordered row of sensory bristles located at the anterior margin of the adult wing. DV boundary cells in the wing disc secrete the Wnt ligand Wingless (Wg), which induces stripes of nearby cells to express proneural genes including senseless (sens) (Giri, 2020).

Each proneural stripe then self-organizes into a periodic pattern of high and low Sens expressing cells. This is orchestrated by two counteracting regulatory loops. The proneural proteins are transcription factors that proportionally stimulate expression of the Notch ligand Delta. Delta activates Notch in neighboring cells and thereby inhibits proneural gene expression in these cells. This generates classic lateral inhibition. At the same time, the proneural proteins co-activate their own transcription within each cell. These interlinked positive feedback loops ensure that initially small differences in proneural protein abundance between neighboring cells evolve into large differences. While sustained and strong expression of Sens is sufficient to drive a cell towards the S fate, neighboring cells downregulate Sens and adopt an epidermal (E) fate (Giri, 2020).

Since lateral inhibition harnesses the variation in proneural protein abundance, this study sought to understand if stochasticity in proneural gene expression is filtered out by spatial signal integration between cells; or transmitted across scales to disrupt ordered sensory bristle patterning. This study has measured the stochastic properties of Sens protein expression and has used experimental perturbations and mathematical modeling to determine the sources of noise. As anticipated, it was discover that molecular birth-death processes and transcriptional bursting influence the stochastic features of Sens expression. Surprisingly, it was found that stochastic features of Sens protein expression are greatly enhanced when one sens allele influences the expression of its paired homolog in trans. When this occurs, cells in the proneural stripes experience abnormally high noise in Sens protein output, which resolves by lateral inhibition into a disordered pattern of sensory bristles. Thus, cis versus trans modes of gene regulation can have major effects on the regularity of sensory pattern formation (Giri, 2020).

An outstanding challenge is to determine whether stochasticity inherent to gene expression is transmitted across scales to vary the fidelity of pattern formation. This study has focused on the organization of sensory bristles along the adult wing margin. During pattern formation, cells experience noise in Sens protein copy number that derives from two sources. One source is from the discontinuous bursts of sens transcription, and the other source is from random birth-death events that affect sens mRNA and protein. For sens, as defined by the 19.2 kb region constituting the transgene, this intrinsic noise was not sufficient to transmit disorder to the adult pattern. However, when sens was subject to trans regulation between paired alleles, protein noise was greatly enhanced, which was sufficient to disorder the adult pattern. Therefore, trans interaction between paired homologs is an unanticipated source of noise. It is tempting to speculate that trans regulation might be a natural means to modulate gene expression noise (Giri, 2020).

Allelic pairing has been observed across multiple organisms. Pairing often precedes trans regulatory interactions, such as X-inactivation. In Drosophila, pairing and trans regulation between homologs has been demonstrated for several developmental genes. Indeed, trans allelic interactions appear to be a pervasive feature of the Drosophila genome. However, pairing of homologous alleles is not necessarily indicative of trans regulation (Giri, 2020).

It remains to be determined how pairing at 57F5 enhances Sens output and noise. The noise profile observed for paired sens alleles at 57F5 can be partly modeled as the effect of enhanced transcription burst size. Since the two-state model uses general rate parameters k_on, k_off, and S_m (the initiation parameter) to regulate bursting, it is agnostic to the specific molecular processes that direct transcription. Distinct mechanisms such as chromatin remodeling, enhancer looping, transcription factor binding-unbinding, or preinitiation complex assembly-disassembly might be rate-limiting for k_on, k_off, or S_m at different levels of Sens expression. Thus, the complex noise profile observed for 57F5 might be a signature of multi-state transcription. Indeed, multi-state transcription kinetics have been observed for other genes. It is also possible that certain types of mRNA state transitions could generate an enhanced profile of protein noise. These might include transitions from an unspliced to spliced state, cytoplasmic mRNA processing, differential translation efficiencies, or toggling between reversible translating and non-translating states. Therefore, although the modeling implicates transcriptional bursting as a major source of protein noise, it remains to be experimentally verified. The use of transgenes inserted into only two sites should be augmented with comparison to more sites and to the endogenous sens gene. Examining transcription bursting via smFISH or MS2-MCP tagged RNA at paired and unpaired loci will be necessary to determine if bursting kinetics are different for different genomic locations (Giri, 2020).

Noise levels differ between 22A3 and 57F5 cells only in the expression regime of 300-800 Sens molecules. Yet, this difference appears to affect pattern order-disorder. It would suggest that a subset of cells with fewer than 800 Sens molecules are at a developmental decision point between E and S (Epidermal and Sensory) fates. This is an order of magnitude less than the approximately 8,000-10,000 Sens molecules observed in terminal S-fated cells. Stochastic fluctuations have the largest impact when protein copy numbers are low. Yet, production of a large number of proteins would raise the time and metabolic cost required to undergo developmental transitions. It suggests that expression noise of certain genes is optimized to allow accurate pattern formation without requiring the production of large numbers of fate-determining proteins (Giri, 2020).

How might this optimization be realized? Individually varying the different rate constants in the two-state model produces very different protein noise-output relationships. Regulating k_on provides the most effective way to increase protein output without increasing noise. Increasing k_on increases the frequency of transcriptional bursts without increasing burst size. Indeed, smFISH experiments show that while sfGFP-sens transcription burst size is constant across the wing margin, mRNA output is regulated by tuning k_on and burst frequency across cells (Bakker, 2020). This study has inferred a similar regulatory mechanism for sens transcription by coupling protein noise measurements to stochastic models (Giri, 2020).

Proper inference of transcription kinetics using protein measurements requires a straightforward correlation between mRNA and protein numbers. Such correlations have been noted in previous work. For instance, bicoid mRNA and protein numbers are reproducible to within 10% and scale proportionately with gene dosage. Stochastic models of transcription and protein production were used to correctly infer mRNA and protein copy numbers for bacteriophage lambda repressor CI and the HIV-1 Long Terminal Repeat promoter . Indeed, protein reporters have been successfully used to infer transcriptional bursting parameters k_on and k_off for a wide variety of transgenic and endogenous mammalian genes (Giri, 2020).

The results suggest that pattern disorder is driven by Sens protein noise rather than protein levels. This might seem counterintuitive since there are many examples of gene overexpression causing developmental phenotypes. The explanation likely lies in the mechanism of wing margin patterning, which occurs in two stages. First, the Wg morphogen induces Sens expression leading to tens to hundreds of protein molecules per cell. Second, Sens expression is self-organized into a periodic row of S and E cells by Delta-Notch mediated lateral inhibition. If all cells should express Sens to an abnormally high level, lateral inhibition still acts on the relative differences between cells to properly resolve the pattern. Consistent with this, reducing endogenous sens gene dose to one copy does not affect bristle pattern formation. On the other hand, enhanced fluctuations in Sens output appear to cause pattern disorder. The simplest interpretation is that during the second stage, cell-to-cell transmission and reception of inhibitory signals is distorted by high intrinsic fluctuations in Sens. If fluctuations are large enough to trigger positive feedback between proneural factors, it would render a cell resistant to lateral inhibition. The net outcome would be cells that spontaneously adopt S fates out of order (Giri, 2020).

Stochastic transcriptional fluctuations have been harnessed in bet hedging systems such as induction of lactose metabolism in E. coli and cell competence in B. subtilis. In mammalian cells, stochastic fluctuations confer phenotypes such as HIV latency periods and acquisition of cancer drug resistance. In developmental contexts, variability in transcription factor output has been shown to affect cell-fate switches that rely on absolute concentration thresholds. However, some developmental systems buffer against expression stochasticity by relying on relative changes in expression or intercellular signaling. This study has shown that a patterning system relying on lateral inhibition can buffer against tissue-scale changes in protein output, but is sensitive to stochastic fluctuations in protein copy numbers (Giri, 2020).

Transcriptional Regulation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Polycomb group genes are required to maintain a binary fate choice in the Drosophila eye

Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, R7 photoreceptor neuron development has been examined in the fly eye . R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp) , as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens) , as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). PcG genes were removed specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s. This study shows that loss of the PcG genes Sce , Scm , or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. This fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, this study shows that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate. It is concluded that cells can use PcG genes specifically to maintain a subset of their binary fate choices (Finley, 2015).

The GMR-FLP/MARCM system allowed allowed the removal of Sce and Scm function specifically from post-mitotic R1/R6/R7 precursors, allowing these genes' roles to be probed in the limited number of binary fate choices that R1/R6/R7 precursors face. In order to adopt the R7 fate, these precursors must choose to: (1) become neurons in response to high RTK activity-they would otherwise become non-neuronal cells; (2) fail to express Svp in response to N activity-they would otherwise become R1/R6s; and (3) fail to express Sens in response to N activity-they would otherwise become R8s. Loss of Sce or Scm from R7s specifically was found to compromises maintenance of the last of these choices. By contrast, no evidence was found that PcG genes maintain either of the other two choices. Sce mutant R7s were examined throughout larval and pupal development and none were found none misexpressed Svp, nor were Sce or Scm mutant R7s that displayed other R1/R6 characteristics found, such as large rhabdomeres positioned at the periphery of the ommatidium or expression of the R1-R6-specific rhodopsin Rh1. While loss of the Abelson kinase was recently shown to cause R neurons to lose expression of the neuronal marker Elav and switch to a non-neuronal pigment cell fate, this study found that Sce and Scm mutant R1/R6s and R7s maintain expression of Elav and the photoreceptor-specific protein Chaoptin, indicating that their commitment to a neuronal fate is also independent of PcG gene function. It is concluded that R7s use Sce and Scm to maintain repression of one but not all alternative binary fate choices (Finley, 2015).

The Sens-encoding region is bound by Pc in Drosophila embryos and by Sce in Drosophila larvae , suggesting that Sens is directly regulated by these proteins in at least some cell types. However, because of the technical difficulty in isolating sufficient quantities of chromatin specifically from R7 cells, it was not possible to determine whether PcG proteins bind the Sens locus in R7s. It remains possible, therefore, that Sce, Scm, and Pc maintain Sens repression indirectly in R7s-however, the evidence suggests that they do so independently of their canonical role in repressing Hox genes (Finley, 2015).

Considerable differences were observed in the strengths of the R7 defects caused by loss of Sce, Scm, Pc, or Psc. One possibility is that these proteins do not contribute equally to PRC1's gene-silencing ability. Indeed, the fly genome contains a second Psc-related gene that plays a redundant role with Psc in some cells, possibly accounting for the lack of defect in Psc mutant R7s. Alternatively, the different wild-type PcG proteins may perdure to different degrees within the mutant R7 clones (the cells that divide to generate the mutant R1/R6/R7 precursors contain a wild-type copy of the mutant gene). Attempts were made, but it was not possible to measure the time course of Sce and Scm protein levels in Sce and Scm mutant R7s, respectively, to test their perdurance directly. However, this thought that perdurance is likely, as this study found that Gal80 perdures until early pupal development within GMR-FLP/MARCM-induced R7 clones (Finley, 2015).

Sce and Scm were found to be required to maintain Sens repression in R7s generated either in the presence or absence of N activity. What might be regulating the deployment of Sce and Scm in these cells? One possibility is that Sce and Scm repress Sens in R1/R6/R7 precursors by default, since these cells never normally express Sens. However, it was found that neither Sce nor Scm is required to maintain the repression of Sens that is established by Svp. Alternatively, Sce and Scm may be deployed to repress Sens as part of a cell's initial commitment to the R7 fate. As mentioned above, wild-type Sce or Scm protein is likely to perdure in newly created homozygous Sce or Scm mutant R7s, respectively, leaving open the possibility that these genes are required not only for the maintenance but also for the establishment of the R7 fate. Previous work showed that the NF-YC subunit of the heterotrimeric transcription factor nuclear factor Y (NF-Y) is also required to maintain Sens repression in R7s. Like the PcG proteins, NF-YC is broadly expressed in all photoreceptor neurons and is not sufficient to cause R7s to adopt R8 fates, indicating that NF-YC is not responsible for the specific role of PcG proteins in R7s. However, the resemblance between the R7 defects caused by loss of Sce, Scm, and NF-YC suggests that NF-Y may participate in PRC1 function. In support of this possibility, loss of the NF-YA subunit from Caenorhabditis elegans also causes defects similar to those caused by loss of the PcG gene sop-2, including derepression of the Hox gene egl-5 (Finley, 2015).

PcG proteins have been shown to silence many regulators of development in addition to their canonical Hox targets, suggesting that PcG proteins are likely to play broad roles in maintaining cell fate commitments. However, whether PcG proteins are used to maintain specific binary fate choices as cells differentiate is unclear. In fact, the opposite is true during stem cell differentiation, when the repression of terminal differentiation genes by PcG proteins must instead be relieved. This paper has have identified a role for PRC1-associated PcG proteins in maintaining a specific binary fate choice made during adoption of the R7 fate-a choice that does not involve Hox gene regulation or misregulation. The same PRC1-associated proteins are not required to maintain two other binary fate choices that R7s must make. It is concluded that PcG genes are indeed used to maintain some though not all binary fate choices (Finley, 2015).

Targets of Activity

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Post-transcriptional regulation; miR-9a targets senseless

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

miR-9a minimizes the phenotypic impact of genomic diversity by buffering Senseless

Gene expression has to withstand stochastic, environmental, and genomic perturbations. For example, in the latter case, 0.5%-1% of the human genome is typically variable between any two unrelated individuals. Such diversity might create problematic variability in the activity of gene regulatory networks and, ultimately, in cell behaviors. Using multigenerational selection experiments, this study found that for the Drosophila proneural network, the effect of genomic diversity is dampened by miR-9a-mediated regulation of senseless expression. Reducing miR-9a regulation of the Senseless transcription factor frees the genomic landscape to exert greater phenotypic influence. Whole-genome sequencing identified genomic loci that potentially exert such effects. A larger set of sequence variants, including variants within proneural network genes, exhibits these characteristics when miR-9a concentration is reduced. These findings reveal that microRNA-target interactions may be a key mechanism by which the impact of genomic diversity on cell behavior is dampened (Cassidy, 3013).

An outstanding question is whether cell behaviors are actively made insensitive to the immense genomic diversity that exists between individuals. This study presents one mechanism by which this may be achieved, and this mechanism was experimentally validated in the context of a natural biological system. Regulation of the transcription factor Sens by miR-9a renders cell fate less sensitive to the varied genomic landscapes of individuals. This effect can be explained by the ability of miR-9a to create a threshold response. Because miR-9a creates a threshold concentration of sens transcript that must be crossed before cell fate is switched, genomic variants that perturb sens transcription are rendered ineffective if the threshold is not passed. If miR-9a is less effective at attenuating sens, then these same variants would be more likely to trigger the cell fate switch. Several variants were found to affect genes that regulate sens transcription, and these variants correlate with a greater impact on the cell fate switch. The variants that affect these genes are not likely sufficient to perturb the network because a constellation of other loci also showed signs of influence. Each of these might have weak and probabilistic effects on sens transcription, which in combination can potentially perturb the network. The impact of such probabilistic fluctuations would be suppressed by the miR-9a-engineered threshold (Cassidy, 2013).

This study is the first demonstration of a molecular mechanism that buffers genomic diversity via gene expression. Transcription factors have previously been invoked as buffering agents of genome variation, although how they achieve this is not known. Their impact on buffering can be significant, as witnessed in classic selection experiments with the sc¹ mutation. Impaired Ac/Sc activity generates increased h², a result that is recapitulated here. Genome buffering can also occur by posttranslational means that involve protein chaperones (Cassidy, 2013).

Although miRNAs have features that theoretically make them suitable for buffering, clearly, this function is not generalized to all miRNAs in all cells. miR-9a shows this function, whereas miR-7 does not. Yet, both miRNAs are highly conserved from fly to human, and the tissue specificity of their expression is also highly conserved. It is proposed that the difference can be found in each miRNA's target within the fate switch network. miR-7 indirectly activates ac/sc transcription, and in turn, its transcription is activated by Ac/Sc. Thus, genome-induced fluctuation of Ac/Sc activity would be amplified and not dampened by miR-7. It could be that miR-7 functions in a nonhomeostatic way in this particular network (Cassidy, 2013).

It is rare for genome variants to have deterministic effects on outcome, and yet the personal genome is being heralded as a new instrument for predicting disease risk and prognosis. Genome variation that correlates with certain outcomes could become a powerful predictor for prevention and treatment. However, counteracting this relationship will be buffering mechanisms that resemble the one is described in this study. The strength and efficacy of such mechanisms will affect the probability that certain variants are disease causative. Buffering mechanisms can also affect the evolution of diseases, in particular cancer. Cancers evolve by clonal expansion, genome instability, and clonal selection It is suggested that tumor heterogeneity is not only manifested by clones of cells with distinct genomes but by the variable buffering of these genomes within a tumor. In support, it has been found that individual tumor cells from a common genetic lineage are phenotypically heterogeneous with respect to growth and responsiveness to therapy. This phenotypic heterogeneity then enables selection for heritable features that promote cell survival and growth in the evolving environment of the tumor (Cassidy, 2013).

The subtle change in miR-9a copy number had large effects on buffering genomic diversity. Hence, the fluid variation in gene copy number, commonly seen in tumor cells, might have an impact in ways previously unforeseen. Moreover, epigenetic variation of gene expression in tumor cells could have similar consequences. In this regard, hypermethylation of the human miR-9 promoter is frequently observed in various carcinomas; this leads to reduced expression of the miRNA. For renal carcinoma, the epigenetic modification is associated with increased risk for recurrence. The current results suggest that reduced miRNA gene expression might affect the ability of cancer cells to evolve under natural and therapeutic conditions (Cassidy, 2013).

In conclusion, this study has identified a miRNA that inhibits the potential for genomic diversity to express itself at the level of a cell phenotype. Because selection experiments are applicable to many genes and cell types, this integrated approach should aid in understanding how genomic diversity is buffered in other organisms for traits that include disease risk and prognosis (Cassidy, 2013).

The COP9 signalosome converts temporal hormone signaling to spatial restriction on neural competence

During development, neural competence is conferred and maintained by integrating spatial and temporal regulations. The Drosophila sensory bristles that detect mechanical and chemical stimulations are arranged in stereotypical positions. The anterior wing margin (AWM) is arrayed with neuron-innervated sensory bristles, while posterior wing margin (PWM) bristles are non-innervated. This study found that the COP9 signalosome (CSN; see CSN5) suppresses the neural competence of non-innervated bristles at the PWM. In CSN mutants, PWM bristles are transformed into neuron-innervated, which is attributed to sustained expression of the neural-determining factor Senseless (Sens). The CSN suppresses Sens through repression of the ecdysone signaling target gene broad (br) that encodes the BR-Z1 transcription factor to activate sens expression. Strikingly, CSN suppression of BR-Z1 is initiated at the prepupa-to-pupa transition, leading to Sens downregulation, and termination of the neural competence of PWM bristles. The role of ecdysone signaling to repress br after the prepupa-to-pupa transition is distinct from its conventional role in activation, and requires CSN deneddylating activity and multiple cullins, the major substrates of deneddylation. Several CSN subunits physically associate with ecdysone receptors to represses br at the transcriptional level. A model is proposed in which nuclear hormone receptors cooperate with the deneddylation machinery to temporally shutdown downstream target gene expression, conferring a spatial restriction on neural competence at the PWM (Huang, 2014: PubMed).

Protein Interactions

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

During development, proneural transcription factors of the bHLH family are required to commit cells to a neural fate. In Drosophila neurogenesis, a key mechanism promoting sense organ precursor (SOP) fate is the synergy between proneural factors and their coactivator Senseless in transcriptional activation of target genes. This study presents evidence that post-translational modification by SUMO enhances this synergy via an effect on Senseless protein. Senseless is a direct target for SUMO modification, and mutagenesis of a predicted SUMOylation motif in Senseless reduces Senseless/proneural synergy both in vivo and in cell culture. It is proposed that SUMOylation of Senseless via lysine 509 promotes its synergy with proneural proteins during transcriptional activation, and hence regulates an important step in neurogenesis leading to the formation and maturation of the SOPs (Powell, 2012).

SUMO enhances SENS's ability to promote proneural activity in reporter gene assays and to promote neurogenesis in vivo. The data suggest that SUMO modification promotes proneural gene autoregulation and is also likely to be important in the regulation of downstream proneural target genes. SUMOylation has a positive effect and deSUMOylation a negative effect on transcriptional activation by proneural/DA/SENS ternary complexes in S2 cells. In contrast, no effect was observed on proneural protein activity in the absence of SENS, suggesting that SENS is the target for SUMO. This is supported by the interactions between SUMO and SENS in the HeLa cell relocalisation and yeast two-hybrid assays, the direct covalent interaction between SENS and SUMO detected in S2 cells and the in vitro SUMOylation assay, and the effect of mutating a putative SUMOylation motif in SENS Zn finger 4 (Powell, 2012).

The latter identified a lysine (K509) in the fourth Zn finger as a candidate for a major SUMOylation site in the SENS sequence. Mutation of this lysine to arginine (K509R) resulted in disruption of SUMO-dependent SENS interaction in the HeLa cell assay, a SENS protein refractory to SUMO stimulation in the S2 cell transcriptional assay, and reduced genetic interaction between SENS and SUMO in vivo. Furthermore, evidence from yeast two-hybrid assays and from analysis of S2 lysates for SENSK509R suggested that additional unidentified lysines may also be SUMOylated. Interestingly, the basal transcriptional synergy between SENS and proneural/DA heterodimers observed in S2 cells appears to be largely dependent on endogenous SUMOylation, as the synergy is strongly reduced by ULP1 cotransfection. Consistent with this, proteomic analysis has shown that S2 cells express SUMO, UBC9 and UBA2 (SAE1) proteins (Powell, 2012).

SUMO affects the activity of the proneural/DA/SENS ternary complex. While the evidence suggests that SENS is the target of SUMOylation, the possibility that the other proteins of the complex may also be SUMOylated is not ruled, but at present there is no evidence for this. Notably, the ATO sequence has no ΨKxD/E motifs, while SC has been shown to be unaffected by SUMOylation in a separate study. DA has three potential SUMOylation motifs, but mutation of each of these does not affect proneural/DA/SENS synergy (Powell, 2012).

The evidence suggests that SUMOylation of SENS enhances transcriptional synergy via an effect on the proneural/SENS ternary complex itself. How might SENS SUMOylation mediate this increase in transcriptional synergy? SUMOylation can exert a positive effect on transcriptional activation by various mechanisms including alteration of subcellular localisation and mediation of interaction with transcriptional coactivators or DNA. In the present case, it is suggested either (1) SUMOylation increases the affinity of SENS for the proneural protein heterodimer hence favouring formation of the more transcriptionally active ternary complex, (2) SUMOylation increases the transcriptional activation or DNA-binding ability of the ternary complex perhaps by inducing a conformational change or (3) SUMO simply stabilises SENS. SUMO is known to modulate protein-protein interactions in other systems: for example SUMOylation of RanGAP1 promotes binding of RanB2 either by creating or exposing a binding site, while NMR studies have provided direct evidence of a SUMOylation-induced conformational change in Thymine-DNA glycosylase. The identified SUMO site of SENS (K509) is within the fourth Zn finger. This is significant because the Zn finger has been shown to be unimportant for DNA binding by SENS but contributes to the transcriptional synergy mediated by proneural/SENS interaction. It is conceivable therefore that SUMOylation at this site increases the affinity of SENS for proneural/Da heterodimers. This would be similar to the proposed enhanced interaction between the TEA family transcription factor Scalloped and its coactivator Vestigial upon SUMOylation of the latter (Powell, 2012).

A major effect of SENS (and therefore SUMO) in promoting SOP specification appears to be via promoting proneural/DA activation of autoregulatory enhancers. This proneural/SENS autoregulatory synergy is thought to have an important role in bypassing the negative regulatory effects of the Hairy/E(SPL) (HES) bHLH repressor proteins downstream of Notch signalling. It is interesting to note therefore that another role for SUMOylation in SOP specification has recently been identified in relation to HES repressors. A model has been proposed in which the repressive activity of HES proteins during neurogenesis (as well as segmentation and sex determination) is disrupted by the SUMO-targeted Ubiquitin ligase (STUbL), Degringolade (DGRN). DGRN binds to SUMOylated Groucho (GRO), the corepressor of HES. This interaction, as well as ubiquitination of the HES proteins, is thought to disrupt the HES-GRO interaction, leading to increased neurogenesis. Hence these two SUMO-dependent mechanisms (i.e. increased SENS coactivation and decreased HES repression) may work in a complementary manner to enhance neurogenesis. It will be important to determine how SOP-specific SUMOylation is regulated in order to elucidate the developmental mechanisms involved (Powell, 2012).

As well as acting as a proneural coactivator, SENS directly represses some target genes via binding to S box motifs. It is therefore conceivable that SUMO can relieve SENS repression of its targets by its promotion of ternary proneural-SENS formation, effectively sequestering SENS from binding to its target S boxes. For example, SENS directly represses the SOP-specific gene, rhomboid (rho), activation of which is crucial for the EGFR-dependent recruitment of secondary SOPs during neurogenesis. ATO indirectly activates rho expression in larval abdominal SOPs by binding SENS and preventing it from binding and repressing the rho enhancer. If for example SUMOylation enhances SENS binding to ATO, then it may play a role in activation of rho and other direct targets of SENS repression (Powell, 2012).

SENS belongs to the GPS (Gfi1/Pag-3/SENS) family of proteins and its mammalian orthologues are the oncogenes, Gfi1 and Gfi1b. The Gfi proteins differ from SENS in containing transcriptional repression SNAG domains near their N-termini, and Gfi1 and Gfi1b have been 03 reported to act mainly as transcriptional repressors. Despite these differences, in the mammalian peripheral nervous system Gfi1 functions in close connection to proneural factors. For example it works in concert with Atoh1 (the mammalian homologue of ATO) in the specification of inner ear hair cells. Gfi1 also has a crucial role in formation of retinal ganglion cells in the mammalian eye, working downstream of a different ATO homologue, Atoh7. Gfi1 also has key developmental roles in the lung and intestine, working together with the mammalian AC/SC homologue Ascl1 in pulmonary neuroendocrine cell production and with Atoh1 in the production of secretory cells of the intestine (Powell, 2012).

It has been suggested that Gfi1 and the mammalian proneural proteins may act as transcriptional coactivators in a similar way to the Drosophila proteins although direct evidence for this is lacking. If corroborated, such interactions could conceivably be modulated by SUMO in a similar mechanism to that which has been found in Drosophila. Interestingly, this is supported by the observation that, like SENS, Gfi1 associates with SUMO pathway proteins including the SUMO-conjugating enzyme UBC9 in a yeast two-hybrid assay although no other evidence has so far been reported for SUMOylation of Gfi1. While SENS has four C-terminal Zn fingers, 19 Gfi has six. The sixth Zn finger of Gfi1 is not needed for DNA binding, and is equivalent to the fourth Zn finger of SENS. This is the location of the putative SUMOylated lysine (K509) in SENS which is completely conserved in the context of the SUMOylation motif in Zn finger 6 of Gfi1. In conclusion, it is possible that Gfi1 activity is modulated by SUMOylation, and this could have an effect via a molecular mechanism similar to that which have been identified for SENS (Powell, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

To determine where the Lyra/sens gene is expressed, in situ hybridization experiments were carried out. Sens mRNA is first expressed in stage 10 embryos in small clusters of 2-4 ectodermal cells. One of the cells in each cluster is the presumptive anterior or posterior sensory organ precursor. This expression quickly refines to ectodermal cells that will give rise to SOP A and P cells. The message then rapidly accumulates in many SOPIs and IIs during stage 11. The mRNA is most abundant in the SOPI and barely detectable or absent in the progeny of the SOPII. During germ band retraction, the remaining SENS mRNA disappears, and by stage 13 the mRNA is only detected in the salivary glands. These observations suggest that sens is most actively transcribed in the SOPI and that other cells of the PNS inherit the message or transcribe the gene at significantly lower levels than SOPIs (Nolo, 2000).

Staining of embryos with anti-Sens antibodies shows a similar expression pattern to the mRNA. With exception of salivary glands and a few ectodermal cells, the protein is confined to proneural fields of some SOPs, nuclei of SOPs, and differentiating cells of the PNS. Protein expression is initiated during stage 10, is maximal during stages 11 and 12, and begins to vanish in differentiated embryos. Double-labeling experiments with P element enhancer detector A37 show that almost all SOPs that express lacZ in A37 embryos express Sens. In contrast to the mRNA, the protein is present in differentiating cells of the PNS. All EMS-induced mutations express the protein, but the levels are either severely reduced or the protein fails to localize to the nucleus in mutants. In summary, Sens is a nuclear protein whose expression is almost exclusively restricted to precursors and early differentiating cells of the PNS (Nolo, 2000).

Larval

To establish if Lyra/Sens is expressed in imaginal tissues, discs were stained with the anti-Sens antibody. Sens is expressed in eye-antennal discs in the R8 photoreceptors, two small clusters of cells in the lateral portion of the disc, and the chordotonal organs of Johnston organs. This pattern is similar to that reported for the A101 enhancer detector (a SOP marker). In leg discs, Sens is expressed in the precursors of the femoral chordotonal organ, as well as in other external sensory SOPs. In wing discs, Sens expression is very dynamic. Sens is first expressed in some ectodermal cells surrounding the cells that will become fated as SOPs. The expression levels in these ectodermal cells are much lower than in the SOPs, and the protein is not confined to the nuclei. This is most easily illustrated for SOPs of the bristles of wing margins but is also observed in other areas. Individual cells within these clusters, the presumptive SOPs, start to accumulate higher levels of Sens protein. Double labeling for anti-ßgalactosidase and anti-Sens in wing discs that carry the A101 SOP marker reveals that the onset of Sens expression precedes that of ßgalactosidase. Both ßgalactosidase and Sens are often coexpressed, except in the SOPs of the bristles of the posterior wing margin where Sens is expressed prior to A101 lacZ. It is likely that no proneural gene is expressed in the noninnervated bristles of the posterior wing margin, although they are similar to other es organs (Nolo, 2000).

Binary cell fate decisions and fate transformation in the Drosophila larval eye

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. This study shows that interplay of two transcription factors Senseless (Sens) and homeodomain transcription factor Hazy [PvuII-PstI homology 13, Pph13] control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. These findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner (Mishra, 2013).

In the larval eye, determination of primary or secondary precursors to acquire either Rh5-PR or Rh6-PR identity depends on the transcription factors Sal, Svp and Otd. Primary as well as secondary precursors have the developmental potential to express Rh5 or Rh6. During differentiation, a pulsed expression of Sens acts as a trigger to initiate a distinct developmental program: Sens acts genetically in a feedforward loop to inhibit the Rh6-PR cell-fate determinant Svp and to promote the Rh5-PR cell-fate determinant Sal. Similarly, in the adult retina, differentiation of 'inner' PRs R7 and R8 requires sens and sal. Sal is necessary for Sens expression in R8-PRs and misexpression of Sal is sufficient to induce Sens expression in the 'outer' PRs R1-R6 (Mishra, 2013).

Svp is exclusively expressed in R3/R4 and R1/R6 pairs of the outer PRs in early retina development. Initially, Sal is expressed in the R3/R4 PRs in order to promote Svp expression. Later, Svp represses Sal in R3/R4 PRs in order to prevent the transformation of R3/R4 into R7. Similarly in larval PRs Svp is repressing Sal in secondary precursors (Mishra, 2013).

Intriguingly, in R8 development in the adult retina Sens also provides two temporally separable functions: First, during R8 specification, lack of Sens in precursors results in a transformation of the cell into R2/R5 fate; second, during differentiation, Sens counteracts Pros to inhibit R7 cell fate and promotes R8 cell fate. Thus, Sens is an early genetic switch in R8-PRs and larval Rh5-PRs that represses an alternate cell fate (Mishra, 2013).

The lack of Sens results in a larval eye composed of only Rh6-PRs. Thus, the default state for both primary and secondary precursors is to differentiate into Rh6-expressing PRs. Rh6 is also the default state in adult R8 PRs: In the absence of R7 PRs (e.g. sevenless mutants) that send a signal to a subset of underlying R8 PRs, the majority of R8 PRs express Rh6. Thus, the genetic pathway initiated by the Sens pulse ensures that primary precursors choose a distinct developmental pathway by repressing the Rh6 ground state. The mechanisms that initiate and control this pulse of Sens remain to be discovered (Mishra, 2013).

In larval PRs as well as in the formation of sensory organ precursors (SOP) in the wing, Sens functions as a binary switch between two alternative cell fates. In the larval eye, this switch occurs when Sens is expressed in one cell type and not in the other. However, during wing disc development the cell fate choice in SOP formation is controlled by the levels, and not the presence or absence of Sens expression: high levels of Sens act synergistically with proneural genes to promote a neuronal fate, while in neighboring cells, low levels of Sens repress proneural gene expression, thereby promoting a non-SOP fate. Thus, Sens uses distinct molecular mechanisms to act as a switch between Rh5 versus Rh6-PR cell fate and SOP versus non-SOP cell fate (Mishra, 2013).

Transcription factors regulate developmental programs in a context- dependent fashion. An example is Sens, which has distinct functions in BO and eyelet development. First, during embryonic development, Sens acts as a key cell fate determinant by regulating transcription factors controlling PR-subtype specification. Second, during metamorphosis Sens inhibits ecdysone-induced apoptotic cell death. Third, in the adult eyelet Sens promotes Rh6 expression. Interestingly, the pro-survival function of Sens appears to be a conserved feature of Sens in other tissues and also in other animal species. In the salivary gland of Drosophila, Sens acts also as a survival factor of the salivary gland cells under the control of the bHLH transcription factor Sage. pag-3, a C.elegans homolog of Sens is involved in touch neuron gene expression and coordinated movement (Jia, 1996; Jia, 1997). Pag-3 was shown to act as a cell-survival factor in the ventral nerve cord and involved in the neuroblast cell fate and may affect neuronal differentiation of certain interneurons and motorneurons. In mice, Gfi1 is expressed in many neuronal precursors and differentiating neurons during embryonic development and is required for proper differentiation and maintenance of inner ear hair cells. Gfi1 mutant mice lose all cochlear hair cells through apoptosis, suggesting that its loss causes programmed cell death (Wallis, 2003). Taken together, these findings support that Sens and its orthologs function in cell fate determination and cell differentiation both in nervous system formation, but also play an essential role in the suppression of apoptosis (Mishra, 2013).

Hazy plays distinct roles in larval PRs and during metamorphosis. First, Hazy is essential during embryogenesis for proper PR differentiation. This early function of Hazy is essential for PRs to differentiate properly during embryogenesis, to express Rhodopsins and to subsequently maintain Rhodopsin expression during larval stages. This function of Hazy is similar to its role in rhabdomere formation in adult PRs and subsequent promotion of Rh6 expression, although it is not required for Rh5 in the adult retina. It is likely that Hazy exerts this function by binding to the RCSI site of the rhodopsin promoters, as has been suggested for the adult retin. Second, during metamorphosis Hazy is required in Rh6-PRs to repress sens, thus allowing these cells to undergo apoptosis. This highlights the reuse of a small number of TFs for distinct functions in the same cell type at distinct time points of PR development. How these temporally distinct developmental programs are controlled on a molecular level remains unresolved. It seems likely that the competence of the cell to respond to a specific transcription factor changes during development (Mishra, 2013).

rh5 and rh6 are expressed in different PRs at different developmental stages: rh5 is expressed in the larval eye and in the adult retina, whereas rh6 is expressed in the larval eye, the adult eyelet and the adult retina. However, the gene regulatory networks controlling rhodopsin expression are distinct in these organs. In the adult retina, a bi-stable feedback loop of the growth regulator melted and the tumor suppressor warts acts to specify Rh5 versus Rh6 cell fate, respectively, while in the larva, Sens, Sal, Svp and Otd control Rh5 versus Rh6 identity whereas Hazy has been shown to maintain Rhodopsin expression. A third genetic program acts downstream of EcR during metamorphosis in Rh5-PRs to switch to Rh6, which requires Sens (Mishra, 2013).

An intriguing question is how the developmental pathways to specify Rh5- or Rh6-cell fates converge on the regulatory sequences of these two genes. It seems likely that parts of the regulatory machinery acting on the rh5 and rh6 promoters are shared between the larval eye, adult retina and eyelet, especially as short minimal promoters are functional in all three different contexts. Future experiments will show how the activity of the identified trans-acting factors is integrated on these promoters to yield context-specific outcomes (Mishra, 2013).

EFFECTS OF MUTATION

Two mutations, sens^M256 and sens^I235 (Salzberg, 1997) fail to complement Lyra¹, a deletion [Df(3L)Ly¹, 70A2-3;A5-6]. Although Lyra¹ embryos are homozygous lethal, a single copy of the Lyra¹ mutation causes a dominant loss of the anterior and posterior wing margin. An EMS screen over the Lyra¹ deficiency permits isolation of other alleles of sens: E1, E2, E53, E54, E58, and E69 (H. Irick, personal communication to Flybase, cited in Nolo, 2000). Most alleles cause similar phenotypes, but much of the phenotypic analysis is carried out with allele E2 (Nolo, 2000).

Since none of the sens alleles causes a dominant wing phenotype, it is conceivable the phenotype associated with Lyra¹ may map elsewhere. However, the following data support the notion that Lyra mutations correspond to gain-of-function mutations of sens. (1) The dominant phenotype associated with Lyra¹ could not be recombined onto a sens mutant chromosome, indicating that both mutations map at the same site. (2) An X ray-induced revertant of Lyra^SX67, a Lyra allele that complements all sens mutations, is homozygous lethal and fails to complement all sens alleles. (3) Lyra mutations have breakpoints in or near the sens gene. (4) Lyra mutations cause ectopic expression of sens precisely in those portions of wing imaginal discs that normally give rise to tissues that are lost in Lyra mutants. (5) Additional copies of Lyra⁺ in Lyra mutants do not alter the wing phenotype (Abbott, 1990). These and other data provide strong evidence that Lyra mutations cause their phenotype by ectopically expressing sens (Nolo, 2000).

To determine the phenotypic consequences of the loss of Sens protein function in imaginal discs, a clonal analysis was performed with the sens^E2 mutation using gamma irradiation and the Minute technique, as well as the FRT/FLP technique. The phenotypes of sens^E2/sens^E2 and sens^E2/sens^1228/4 embryos are similar, suggesting that sens^E2 is a severe loss of function or null allele (sens^E2 contains a stop codon in the middle of the coding sequence). In addition, sens^E2/sens^E2 embryos and flies can be fully rescued by the transgene, showing that this chromosome does not carry other obvious viable or lethal mutations. Mutant clones lack bristles, sockets, and microchaetae. This loss of bristles and sockets is confined to the mutant cells, indicating a cell-autonomous requirement for sens (Nolo, 2000).

In imaginal discs, sens^E2 clones contain low levels of mutant cytoplasmic Sens protein several hours prior to puparium formation. However, they contain undetectable levels of Sens protein at and after puparium formation. Mutant sens^E2 clones are marked by the absence of ßgalactosidase driven by the arm-lacZ transgene. After puparium formation, mutant clones in imaginal discs fail to express the Scute protein. However, mutant clones in less mature wing discs do contain Scute protein in proneural clusters, showing that Sens is not required for Scute expression in ectodermal cells. In addition, some newborn SOPs in mutant clones contain higher levels of Scute than surrounding cells of the proneural field. However, these are consistently lower levels of Scute than in neighboring SOPIs. The levels of Scute in mutant SOPIs remain low and are not maintained. No Scute expression is observed at puparium formation. It is concluded that Sens protein is required to further enhance and maintain expression of Scute in SOPs (Nolo, 2000).

Thus, clonal analysis shows that loss of sens during imaginal disc development leads to loss of bristles, socket cells, and neurons. Unlike in embryos, this loss is paired with a loss of all markers tested in SOPs or their daughters (Asense, Scute, Cut, mAb22C10, and Prospero). This phenotype is more severe than in embryos, suggesting a more prominent requirement for Sens in larval SOPs than in embryos. The phenotype associated with loss of sens in wing imaginal disc is similar to the phenotype associated with the loss of scute and achaete. In addition, clonal analysis indicates that sens is required in cells in which it is expressed. It is therefore conclude that sens is a cell autonomous factor required for an early event in PNS differentiation. Unlike the proneural genes, the requirement for sens is not restricted to a type or subpopulation of sensory organs; rather, it is required for most PNS organs and cells (Nolo, 2000).

The specification of external sensory organs of anterior wing margin has been shown to be dependent not only on Scute and Achaete, but also on Asense. Therefore, SOPs of anterior wing margin were stained with anti-Asense. Asense is not expressed in mutant SOPs, showing that Asense expression is also dependent on Sens. No Cut expression was observed in the SOPIIs, although expression of Cut in other cells of the margin is unaffected. Similarly, expression of the antigen recognized by mAb22C10 in early differentiating cells of mutant clones is absent. In summary, these data suggest that SOPIs may at least in part be specified in sens mutant clones because some express higher levels of Scute than the other cells of the proneural cluster. However, loss of Sens activity then causes a failure to further upregulate and maintain proneural gene expression leading to cell loss in the adult imago (Nolo, 2000).

Effects of mutation: Lyra functions in the wing

Like Lyra, many mutations that affect wing morphogenesis have mutant phenotypes in which there are missing sectors of the wing margins. These include vestigial, Notch, Delta, cut, apterous, and others, which have been shown to play important roles during fruitfly development. It is known that many of these so-called 'wing scalloping' mutations, including cut and vestigial, are caused by excessive cell death in the prospective wing margins of late larvae following a period of apparently normal development. In Lyra mutants, although there is a significant reduction (10%-20%) of the number of cells in the adult wing, no evidence of apoptotic or necrotic cell death was found by transmission electron microscopy, acridine orange, or trypan blue staining in third instar and pupal discs. These and other data have led to the suggestion that Lyra may affect more fundamental parameters of cell growth and specification. The Lyra mutant has therefore been of interest to those investigating wing margin development (Nolo, 2001).

The Lyra¹ mutation is associated with an X-ray-induced deletion uncovering cytological bands 70A2-3;A5-6. It is a dominant mutation that causes a regular and predictable pattern of loss of the anterior and posterior wing margins along with a small amount of nearby wing surface tissue. The presence of a duplication of the chromosomal region carrying the wild-type Lyra locus in a Lyra¹ background does not suppress the Lyra phenotype (Abbott, 1990). Hence, the dominant phenotype is not due to haploinsufficiency. This suggests that Lyra is a gain-of-function mutation and is likely to be a neomorphic allele characterized by spatial and/or temporal misregulation of expression of a gene product. In addition, the results of clonal analysis with wild-type clones in Lyra/+ flies indicate a non-cell-autonomous function (Abbott, 1986), suggesting that the Lyra mutation may affect processes requiring cross-talk among cells, such as specification of positional information or lateral inhibition (Nolo, 2001).

Properties of the Lyra¹ phenotype have been studied extensively by Abbott (1986, 1990). Five key observations and conclusions follow: (1) excessive cell death in the putative wing margins of third instar and early pupal wing discs does not account for margin loss in Lyra adult wings; (2) although Lyra mutants do not form anterior and posterior wing margins, there is a normal dorsal/ventral compartment boundary as well as a zone of nonproliferating cells (ZNC) along the 'de facto' Lyra wing margin; (3) a monoclonal antibody, which binds the unidentified E1C antigen expressed in the larval wing margin precursor, allows for the demonstration that an effect of Lyra on anterior and posterior wing margins is apparent early in the third larval instar; (4) margin rescue experiments using clonal analysis show that wild-type Lyra is not required for bristle development per se; (5) further analysis of shape and position of clones indicates that the wing margin, defined as a set of several rows of cells along either side of the dorsal/ventral boundary, plays an important role in wing morphogenesis. This observation presaged the current paradigm that interactions among a number of gene products expressed in the margin region, often acting across the compartment boundary, serve to organize wing development (Nolo, 2001).

Salzberg (1994, 1997) has shown that the senseless gene affects the development of the PNS and report three alleles: two ethyl methanesulfonate (EMS)-induced alleles (sens^M256 and sens^I235) and one induced by P-element dysgenesis (sens^1228/4). The lethality associated with these alleles has been mapped by meiotic recombination to 3-40.5, the same location to which Lyra¹ maps (Nolo, 2000). senseless mutations M256 and I235 failed to complement the lethality of Lyra¹, which is associated with deletion 70A2-3;70A5-6. The Lyra¹ deletion uncovers three essential complementation groups: l(3)70Aa, l(3)70Ab, and l(3)70Ac. Unfortunately, these mutants no longer exist. However, Holly Irick and Peter Cherbas carried out an EMS mutagenesis to identify lethal mutations uncovered by Df(3L)BK10 (71C3;71E5). Because the Df(3L)BK10 chromosome is marked with Lyra¹ it is suspected that some of the Irick and Cherbas lethals are in the Lyra¹ deficiency. Twenty-seven lethal mutants that failed to complement the Lyra¹ Df(3L)BK10 chromosome were tested and 8 lethal mutations were isolated that fail to complement the Lyra¹ deficiency (Nolo, 2001).

Complementation tests for the lethal phenotype show that six of these mutations are alleles of senseless, referred to as complementation group l(3)70Ad in FlyBase. This complementation group is presumably allelic to one of the lost l(3)70A complementation groups. The sens alleles were designated E1, E2, E53, E54, E58, E64, E69, and E87. (Irick and Cherbas, personal communication to FlyBase cited in Nolo, 2001). All but E64 and E87 are still available (Nolo, 2001).

The Lyra¹ deficiency in trans to other senseless alleles causes lethality but these mutant embryos do not display a severe loss of neurons as typically seen in homozygous senseless mutations (Nolo, 2000). Indeed, the following observations suggest that the Lyra¹ mutation is not a loss-of-function allele of senseless. (1)Lyra¹/sens mutant embryos display either no loss of PNS neurons or a very subtle loss, indicating that the deficiency associated with Lyra¹ does not result in the lack of the senseless gene product; (2) none of the senseless alleles cause a loss of wing margin phenotype in heterozygous flies (sens/+), indicating that haploinsufficiency of sens does not cause the Lyra phenotype; (3) a second, independently generated dominant allele of Lyra, Lyra^SX67, interacts additively with Lyra¹ to produce a more severe margin loss, but complements all senseless alleles. Since Lyra mutations are dominant and their phenotype is not caused by haploinsufficiency of senseless, they are presumed to be either antimorphic (dominant negative) or neomorphic (gain of function) in nature. An antimorphic nature is most unlikely since duplications of the chromosomal region do not ameliorate the phenotype associated with Lyra¹ (Abbott, 1990). Furthermore, the Lyra¹/sens mutants do not display obvious defects in the PNS, as would be expected if Lyra was a dominant-negative allele of senseless. It is therefore concluded that Lyra mutations are neomorphic mutations (Nolo, 2001 and references therein).

The following data support the neomorphic nature of the Lyra mutations, that is, that they are gain-of-function, regulatory mutations of senseless: (1) the dominant phenotype associated with Lyra¹ could not be recombined onto a senseless mutant chromosome, indicating that both mutations map at the same site and that the Lyra phenotype may be breakpoint dependent; (2) molecular analyses show that the distal breakpoint of Df(3L)Ly¹ affects a genomic fragment that contains the 3' end, including the 3' untranslated region, of the sens gene; (3) an X-ray-induced revertant of Lyra^SX67, Lyra^SX67R12, is homozygous lethal and fails to complement all the senseless alleles, showing that Lyra^SX67 is associated with senseless; (4) both Lyra mutations cause ectopic expression of Senseless in wing imaginal discs (see below). These observations strongly indicate that the Lyra alleles are neomorphic/gain-of-function mutations of senseless (Nolo, 2001).

To demonstrate that ectopic expression of Senseless can mimic the Lyra phenotype, flies were constructed that carried different UAS-senseless transgenes under the control of GAL4 drivers that express GAL4 rather specifically in the wing disc. Most and possibly all GAL4 drivers that cause widespread expression of GAL4 are lethal in the presence of UAS-senseless. Ectopic expression of Senseless in the wing disc using the C1003-GAL4 driver causes a phenotype that is similar to that observed in Lyra mutants: the wing margins are severely affected whereas the rest of the wing is unaffected. Expression of Senseless in a domain that corresponds to the wing margin using the C96-GAL4 driver also causes a loss of wing margin. In this case the loss is not as severe as that induced by the more ubiquitous driver. These observations clearly show that ectopic expression of Senseless is sufficient to cause loss of wing margin tissue. Conversely, they indicate that other areas of the wing disc do not respond to ectopic Senseless expression with tissue loss. Instead, as reported previously (Nolo, 2000), it is consistently observed that ectopic Senseless causes scattered supernumerary bristles on nonmargin surfaces of adult wings. Moreover, ectopic expression of Senseless in wing discs, using a dpp-GAL4 driver, results in large clusters of extra bristles on the notum as well as parts of the wing surface. In leg discs, this driver causes a severe loss of all distal parts of the leg. It is concluded that ectopic expression of Senseless causes very different phenotypes: loss of tissue in some areas of imaginal discs and extra sensory organs in others (Nolo, 2001).

Four markers were tested to determine the effects of Lyra mutations on the expression of key genes that have been shown to play important roles in wing development. The vestigial gene can be viewed as the wing selector gene because its lack of expression causes wing loss and its ectopic expression causes extra wing tissue. Vestigial expression at the dorso-ventral boundary is essential to wing margin development. Furthermore, vestigial is a marker for wing identity and has an important function in wing growth (Nolo, 2001 and references therein).

The effect of both Lyra mutants on the expression pattern of lacZ driven by the vestigial boundary enhancer vgBE was tested. lacZ expression of the boundary enhancer is almost entirely lost in the anterior and posterior portion of the wing pouch of Lyra¹ but is restored in the revertant, which has the same pattern of expression as the wild-type adult wing. The pattern is similar in Lyra^SX67, except that slightly more prospective margin is missing, in agreement with the more severe margin loss in the adult wing. Lyra has no effect on the vestigial quadrant enhancer, vgQE, which controls later Vestigial expression and growth of the nonmargin portion of the wing pouch. In this case Vestigial is expressed throughout the wing blade but not in the prospective margin. Immunocytochemical staining with the anti-Vestigial antibody shows a different pattern of expression in Lyra wing discs in the anterior and posterior area of the wing pouch when compared to wild type. It is not known what underlies this altered pattern, but it may be due to loss of Vestigial expression at the dorso-ventral boundary. Since loss-of-function clones of vestigial (vg^-/vg^-) do not proliferate in the wing, the loss of wing margin tissue in Lyra mutants could be caused by a partial loss of Vestigial expression at the anterior and posterior wing boundary. This in turn may cause loss of cell proliferation during pupal wing development (Nolo, 2001).

In addition to vestigial, wingless has also been shown to play an essential role in wing development. Wingless protein is secreted and is produced in a stripe of three to four cell rows stradling the dorso-ventral boundary. The stripe of Wingless-expressing cells induces neighboring cells to differentiate into the bristles that are present at the wing margin. Removing Wingless in second or early third instars results in the loss of tissue from the wing margin. The role of Wingless with respect to regulation of Vestigial expression at the dorso-ventral boundary is still controversial. However, it is fairly clear that Notch signaling is the primary inducer of vgBE. Hence, Wingless expression in Lyra mutants may provide an independent means to assess the effect of Lyra mutations on wing development. Wingless expression is severely reduced in the anterior and posterior domain of the wing pouch of Lyra mutants. With the exception of the central domain of the dorso-ventral boundary, where Wingless expression is apparently normal (as is the Lyra wing margin), Lyra's expression is confined to a narrow domain in which levels of Wingless protein are reduced severely. Since Wingless is an important secreted factor for wing margin development, this reduction in expression in Lyra mutants may act in an additive fashion with the loss or severe reduction of Vestigial expression (Nolo, 2001).

Given the similarities between the loss of wing margin tissue in some cut and Lyra mutants, Cut expression was examined in Lyra mutants. Cut is expressed in a row that is two to five cells wide at the dorso-ventral boundary. This expression is largely overlapping with that of Wingless and the vestigial boundary enhancer but occurs in the mid-third instar, much later than either Wingless or vgBE. Loss of Cut expression on both sides of the wing boundary results in extensive notching of the margin. cut has been shown to be a direct target of Notch, but not of wingless. In addition, while the initiation of Wingless expression is not dependent on cut, maintenance of Wingless expression is dependent on cut. Cut expression is shown to be essentially abolished in Lyra mutants in the anterior and posterior region of the wing pouch (Nolo, 2001).

Since Notch signaling plays a prominent role in the regulation of the expression of vgBE, wingless, and cut, attempts were made to determine if Notch signaling is affected. Lyra wing discs were stained with anti-Delta antibodies since anti-Notch antibody immunohistochemical staining of wing discs failed because of high background levels. There is an obvious reduction in the expression of Delta in the anterior and posterior wing pouch along the presumptive wing margin. Hence, one of the key known activators of Notch signaling at the dorso-ventral boundary is altered and reduced in its expression pattern. In summary, four known markers that have previously been shown to be required for the development of the wing margin and the rows of bristles along the margin are not expressed properly in Lyra mutants. In addition, the domains of expression that are affected in these mutants correspond to the domains that are affected in Lyra mutant discs and adult wings and are contained within the domains in which Senseless is expressed ectopically. These data suggest that ectopic expression of Senseless in Lyra mutants may be able to downregulate the expression of several genes that play a pivotal role in wing margin development, possibly by downregulating Notch signaling (Nolo, 2001).

To further investigate the ability of Senseless to downregulate the expression of specific genes, the effect of ectopic expression of Senseless on Wingless and Cut expression was examined. Senseless was ectopically expressed using the C96-GAL4 wing margin driver; this was followed by staining with anti-Senseless. Senseless overexpression causes a dramatic downregulation of Wingless and Cut protein levels, although in both cases clusters of immunoreactive cells along the wing margin remain. Similarly, when using the dpp-GAL4 driver to ectopically express Senseless along the anterior-posterior wing boundary, a precise disruption in the continuity of Wingless and Cut expression was found where the dpp stripe is normally expressed. This downregulation correlates with a loss of the distal tip of the wing. Note also that ectopic Senseless expression causes ectopic Cut expression in some cells of the wing pouch that normally do not express Cut, as expected from previous observations (Nolo, 2000). In summary, these data demonstrate that ectopic expression of Senseless in the wing margin is a potent repressor of expression of key players previously shown to function in wing margin development (Nolo, 2001).

The loss of wing margin in Lyra mutants can be viewed as the sum of two components. The first component is an effect on margin determination in the developing wing disc. Indeed, these data are in agreement with numerous observations showing that loss of Notch signaling causes loss of expression in the wing margin of the patterning genes wingless and vestigial. The second component corresponds to an effect on cell proliferation. Indeed, loss and gain of Notch signaling experiments have been shown to cause a severe decrease and increase in cell proliferation, respectively. The data suggest that loss of Delta causes a loss of Notch signal and a loss of cell proliferation in the wing margin. The reduction in cell proliferation in Lyra wing development begins shortly after pupariation and continues during the first half of pupal development (Abbott, 1990). This is the time window in normal development when differentiation of bristles and trichomes takes place as well. To further examine how ectopic Senseless affects wing margin specification and differentiation during Lyra wing development, the expression patterns of Scute and string were studied (Nolo, 2001).

scute is a proneural gene belonging to the achaete/scute complex and a basic Helix Loop Helix (bHLH) transcription factor required for determination of SOPs in the anterior wing margin. A downregulation of Scute expression in the anterior pouch of the wing disc is observed. Indeed, in Lyra^Sx67 wings, there are few SOPs expressing Scute at the anterior wing margin. This is in sharp contrast to ectopic expression of senseless in other epithelial cells of the wing disc where it causes induction of Scute expression (Nolo, 2000 and Nolo, 2001).

Wingless is required for differentiation of bristles late in margin development. Indeed, high levels of Wingless are known to be required for the proper expression of the proneural SOP determinants acheate and scute. In addition, Cut expression in third instar discs has been shown to be dependent on Wingless expression, while Cut is also required for the maintenance of Wingless expression. Since Cut is essential for all wing margin bristles, both innervated and noninnervated, it is proposed that the combined reduction in Wingless and Cut expression in Lyra mutants may cause a secondary reduction in proneural gene expression in the wing margin, as revealed by Scute staining. This reduction in expression should lead to a loss of numerous bristles in the anterior and posterior wing margin. However, these observations do not provide a rationale for the loss of wing blade cells adjacent to the margin, which are also observed in Lyra mutants (Nolo, 2001).

The failure to form SOPs in the wing discs of Lyra mutants predicts that the set of two cell divisions required for differentiation of margin bristles in the early pupa will not take place. The reason for the loss of the surrounding unspecialized margin cells in the adult wings of Lyra is not as obvious, but one hypothesis is that these cells also fail to proliferate. To test this, the mRNA expression pattern of String was examined. String mRNA is normally expressed in the central cells of both the anterior and posterior wing margin during the later third instar larval stage even though margin cells are arrested at this time and cell proliferation does not begin until early pupariation. in situ experiments with string confirmed this expression pattern in wild-type discs. But in Lyra third instar wing discs, the mitosis-inducing phosphatase String (Cdc25) is severely downregulated in the anterior and posterior area of the prospective wing margin, as indicated by in situ hybridization. This is consistent with an overall lack of proliferation in the anterior and posterior margin region. However, it is also possible that the non-bristle-forming cells are present in the wing margin, but that they lose their capacity to flatten and secrete margin elements (trichomes), which serve as the visible hallmark of each cell. This could be caused by their lack of exposure to the sequence of proteins required for determination of the wing margin (Nolo, 2001).

vestigial can be viewed as a 'wing selector' gene, a view that is supported by the observation that its ectopic expression can rescue loss of Wingless. Loss of Vestigial in the wing disc also causes a failure of wing cells to proliferate. It has been proposed that the vgBE is induced by Notch signaling when and where Wingless is active at the developing wing margin. The main function of Wingless is to enforce gene expression in the wing disc rather than to initiate it. Hence, the combined loss of vestigial expression at the boundary and the strong reduction in Wingless expression at the wing margin may affect cell proliferation and cell identity not only in the wing margin, but also in a few cell rows adjacent to the anterior and posterior wing margin. This model is in agreement with the observation that no alterations in the expression pattern of the quadrant enhancer of vestigial are found in Lyra mutants and that Lyra wing discs exhibit a dramatic reduction in string expression in the cells along the dorso-ventral boundary. Since string has been shown to induce mitosis, and since Lyra mutants exhibit no cell death and a loss of cells in pupal development, a causal relationship between these observations is proposed. At the root of the Lyra phenotype may be the observation that the Delta signal is impaired, which should lead to a decrease in Notch signaling. This decrease may explain the loss of Wingless, Vestigial, and Cut expression, which have all previously been shown to depend on Notch signaling. This defect in Lyra mutants is proposed to underlie the effect on margin determination in the developing wing disc and the reduction in cell proliferation in early pupae (Nolo, 2001).

senseless is necessary for the survival of embryonic salivary glands in Drosophila

Apoptosis in developing Drosophila embryos is rare and confined to specific groups of cells. How do salivary glands of Drosophila embryos avoid apoptosis? senseless (sens), a Zn-finger transcription factor, is expressed in the salivary primordium and later in the differentiated salivary glands. The regulation of sens expression in the salivary placodes is more complex than observed in the embryonic PNS. sens expression is initiated in the salivary placodes by fork head (fkh), a winged helix transcription factor. The expression of sens is maintained in the salivary glands by fkh and by daughterless (da), a bHLH family member. Salivary gland-expressed bHLH (Sage), a salivary-specific bHLH protein, has been identified as a new heterodimeric partner for Da protein in the salivary glands. In addition, the data suggest that sage RNAi embryos have a phenotype similar to sens and that sage is necessary to maintain expression of sens in the embryonic salivary glands. Furthermore, in the salivary glands, sens acts as an anti-apoptotic protein by repressing reaper and possibly hid (Chandrasekaran, 2003).

In situ hybridization shows that sens mRNA is first expressed in the dorsal cells of the salivary placodes at stage 11 of embryogenesis. As the embryo undergoes germ band retraction, sens mRNA expression expands to include all the cells of the salivary placodes, but is excluded from the salivary duct precursors.A similar expression pattern is observed for Sens protein, though the protein is not expressed at high levels in the ventral part of the salivary placodes. Though sens mRNA and protein disappear from the embryonic PNS by stage 13, both continue to be expressed in the embryonic and larval salivary glands (Chandrasekaran, 2003).

Embryos mutant for sens^E2 have small salivary glands, about half to a third the size of normal salivary glands. In addition, the salivary glands of stage 16 sens^E2 embryos are smaller than those in stage 13 embryos, suggesting that the loss of cells may be progressive. Similar phenotypes were obtained for two other alleles, sens^E58 and sens^I235, as well as for transheterozygotes of sens^E2 and a deficiency for sens, suggesting that sens^E2 behaves as an amorph in these studies. The phenotype seen in the sens^E2 mutant salivary glands can be rescued by overexpressing sens in the embryo, indicating that the observed phenotype is due to the lack of sens function in the salivary primordium (Chandrasekaran, 2003).

Because the salivary glands are the only non neural tissue in the embryo to express sens, it was of interest to see how different the regulation of sens transcription is in this tissue. In the PNS, Da forms heterodimeric complexes with proneural bHLH proteins. These complexes are necessary for both the initiation and maintenance of sens expression in the sensory organ precursors. The proneural genes achaete, scute, lethal of scute, asense and atonal are mainly expressed in the proneural clusters and are absent from the salivary placodes. By contrast, da expression is ubiquitous in the early embryo and is upregulated in the salivary glands of older embryos, suggesting that da might be involved in regulating the expression of sens in the salivary placodes. If so, da mutants would have a salivary phenotype similar to sens mutants. In confirmation of this hypothesis, salivary glands in da mutants were smaller than in wild-type embryos. In situ hybridization showed that the levels of sens mRNA (and protein) are dramatically reduced in the salivary glands of da mutants, suggesting that Da regulates sens in both the PNS and salivary gland. However, unlike the PNS, salivary gland sens expression initiates in the absence of da (Chandrasekaran, 2003).

Although known Da partners are not expressed during salivary development, a genome-wide survey for genes encoding bHLH proteins identified sage, a gene whose expression is salivary gland-specific in the embryo. The expression of sage in the salivary placodes is first observed at stage 10, the stage at which the first Scr targets begin their salivary expression. sage continues to be expressed in the salivary glands throughout embryogenesis and into larval development. Scr-mutant embryos lack salivary glands and do not express sage. Double stranded RNA interference was used to test whether sage is required for salivary gland development. Forty percent of the embryos injected with sage dsRNA, showed small salivary glands, compared with 10% for the injection buffer control. Sens levels are reduced by sage dsRNA injection. These observations indicate that sage is required for regulation of sens in the salivary glands. Sens expression does initiate in the absence of sage, as it does in da mutant embryos (Chandrasekaran, 2003).

It has been suggested that class II bHLH proteins, the class that includes Sage, can heterodimerize with Da. To test whether Sage indeed forms a complex with Da, a GST pulldown assay was used with ³⁵SDa protein and GST-Sage. Da protein binds to GST-Sage. In addition, Da does not bind to a truncated Sage that lacks the C-terminal bHLH domain. These observations show that Da can partner with Sage in vitro and suggest that Sage and Da form a complex in vivo to regulate the expression of sens in the salivary glands (Chandrasekaran, 2003).

In the sensory organ precursors of the PNS, sens is necessary to maintain the expression of the proneural genes. Similarly, sage RNA is decreased in sens mutants, suggesting a positive feedback loop between sens and sage. However, expression of da appears to be unaffected in sens mutants (Chandrasekaran, 2003).

Although da and sage are necessary for maintaining sens expression, initiation of sens in the salivary placodes did not depend on either of these genes. Since sens expression in the salivary placodes initiates at stage 11, later than primary Scr target genes, it was thought sens might be indirectly activated by Scr through one of these primary targets. As expected, sens expression was found to be absent in Scr mutant embryos. sens expression is unchanged in embryos mutant for several Scr-regulated early transcription factors such as huckebein, trachealess and eyegone. However, fkh mutant embryos show a complete absence of sens expression in the salivary placodes and never express sens at the later stages. The expression of sens in the PNS is unaffected in these mutants. da and sage RNAs were unchanged at stages 10 and 11 in fkh mutants, indicating that the lack of sens is not due to the effects on sage or da expression. There was a slight reduction in sage RNA at stage 12, which may be due to the positive feedback loop between sens and sage in the salivary placodes. Thus, sens expression in the salivary placodes is initiated by fkh and is maintained at high levels throughout embryogenesis by da and sage (Chandrasekaran, 2003).

Thus, the regulation of sens in the salivary glands is more complicated than in the proneural tissues. sens expression in the salivary glands can be divided into two parts: initiation and maintenance. sens is initiated in the salivary placodes in response to fkh, one of the initial set of salivary genes that are directly activated by Scr at the beginning of stage 10 (4.3 hours AEL). sens expression begins about an hour later and may be directly regulated by fkh. There are FKH binding sites present at the 3' end of sens and a fragment carrying these sites is sufficient to recapitulate the expression in the salivary glands (Chandrasekaran, 2003).

Since sens is a fkh target and because both sens and fkh embryos show extensive salivary apoptosis, it was thought that apoptosis in fkh mutants might be caused by lack of sens. Because rescuing cell death in fkh mutants does not rescue normal morphogenesis, it was suggested that sens normally protects salivary cells from cell death, and other fkh target genes direct the cell movements and shape changes needed to form the salivary gland. However, the apoptosis of the salivary placodes in fkh mutants could not be rescued by ubiquitous expression of sens. There are two explanations for this result. The first possibility is that sens was not overexpressed at high enough levels to overcome cell death. However, this is likely not to be the case because the same arm-GAL4:UAS-sens combination was used to rescue the sens phenotype. Furthermore, arm-Gal4:UAS-P35 rescues cell death in sens mutants. Thus, the second possibility is favored, that loss of fkh leads to multiple proapoptotic changes, only one of which is the failure to activate sens (Chandrasekaran, 2003).

Although Fkh can initiate expression of sens in the salivary placodes, both Da and Sage are required for high level sens expression at later stages. Da is also known to control the expression of sens in the PNS. There, it partners with the proteins of the Achaete-Scute Complex or with Atonal to regulate sens expression. For sens regulation in the salivary primordium, a new Da partner, Sage, which belongs to the bHLH proteins of the Mesp family, has been identified. These results are the first to demonstrate the ability of Mesp family members to heterodimerize with Da. It is shown, using RNAi, that absence of sage leads to a decrease in the size of the glands and a reduction in levels of Sens. In turn, Sens appears to positively regulate the levels of sage mRNA in the salivary glands. The existence of this positive feedback loop leads to the question of which protein, Sage or Sens, is the true antagonist of apoptosis in the salivary glands. The presence of sage mRNA in sens mutants sheds some light on this issue. In sens mutants, high levels of Rpr-11-lacZ are induced at stage 12, in the salivary placodes. At this stage, sens mutant embryos still express sage and da mRNA in the placodes at normal levels. Reduction in sage mRNA is not observed until stages 13-14, by which time the salivary glands of sens mutants are already reduced in size. These results indicate that sens, not sage, is necessary to maintain the survival of the salivary gland cells (Chandrasekaran, 2003).

A similar circuit controls the regulation of expression of Gfi1 (Wallis, 2003), the vertebrate ortholog of sens, in the inner ear cells of mice. The bHLH protein Math1, termed Atoh1, a homolog of atonal, is necessary to maintain Gfi1 mRNA, but not for its initiation in the inner ear cells. It would be interesting to examine if fkh family members are involved in this case to initiate the Gfi1 expression. However, the feedback regulation of sens onto sage or proneural genes is not observed between Gfi1 and Math1 (Chandrasekaran, 2003).

Senseless was identified in a genome-wide analyses for transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4²²¹) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4²²¹-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

In contrast to the genes that coordinately affect dorsal dendrite outgrowth and lateral branching/outgrowth, a group of 21 genes (group B) were identified that have opposing effects on dendrite outgrowth and branching, suggesting that dendrite outgrowth and branching might partially antagonize one another. RNAi of 19 of these genes resulted in dorsal overextension of primary dendrites and a reduction in lateral branching/lateral branch extension. In the most severe cases, such as RNAi of the transcriptional repressor snail, dorsal overextension of almost completely unbranched dendrites was found. Like snail(RNAi), RNAi of the nuclear hormone receptor knirps, the transcriptional repressor l(3)mbt, as well as 15 other genes, all caused dorsal overextension of primary dendrites. As in the case of genes that normally limit arborization, RNAi of these genes rarely caused dendrites to cross the dorsal midline (Parrish, 2006).

Proper dendritic routing is important for primary dendrites of ddaD and ddaE to grow in parallel toward the dorsal midline without crossing each other and for secondary branches of ddaD and ddaE to avoid the space between ddaD and ddaE. Therefore, there must be mechanisms that promote this stereotyped arborization pattern, including signals that promote anterior arborization of ddaD and posterior arborization of ddaE, as well as signals that antagonize posterior arborization of ddaD and anterior arborization of ddaE. Indeed, RNAi of 10 TFs disrupted the dendritic routing patterns of ddaD and ddaE, resulting in aberrantly oriented primary dendrites. RNAi of cg1244, bap55 (brahma associated protein of 55kD), cg9104, cg4328, and cg7417 resulted in inappropriate anterior arborization of ddaE as well as inappropriate posterior arborization of ddaD. Anterior or even ventral displacement of ddaD concomitant with anterior arborization of ddaE was also observed as well as displacement of ddaE arbors concomitant with misrouting of ddaD. Finally, reducing sens function by RNAi or genetic mutation caused extensive mixing of dendritic arbors from ddaD and ddaE, in addition to dorsal overextension of primary dendrites and an overall reduction in the number of class I neurons (Parrish, 2006).

In addition to the seven genes that function to restrict class I neuron number and control dendrite morphology, three other genes are required to maintain the number of class I neurons. Reduction of their function caused a reduction of class I neurons and defects in dendrite morphogenesis in the remaining neurons. For example, RNAi of the zinc finger TF senseless (sens) reduced the number of class I neurons, consistent with previous findings that sens is required for development of most cells in the PNS. In addition, sens(RNAi) or a sens loss-of-function mutation caused an increase in dendrite outgrowth and mixing of dendrites in segments with both ddaD and ddaE present. Similarly, RNAi of the proneural bHLH TF atonal (ato) reduced the number of class I neurons, consistent with previous findings that chordotonal organs and some md neurons are absent in embryos lacking ato. Consistent with reports that ato functions in neurite arborization in the larval brain, it was also found that ato(RNAi) caused altered arborization patterns of class I dendrites. Thus, it is likely that multiple TFs that regulate neuron number also regulate aspects of post-mitotic neuronal differentiation (Parrish, 2006).

Since group A and B TFs regulate aspects of dendritic growth and branching, potential epistatic relationships among TFs was explored in these phenotypic classes. To do this, RNAi was used to knockdown expression of select TFs in Drosophila embryos carrying a loss-of-function mutation in either the group B/C gene senseless (sens) or the group A gene abrupt (ab). sens mutant class I dendrites overextend dorsally and have reduced lateral branching in addition to routing defects. In sens mutants, RNAi of the group A genes Su(z)12 and ab, which cause increased lateral branching following RNAi in wild-type embryos, led to an increase in lateral branching compared with injected controls. Therefore, Su(z)12 and ab function are still required to limit arborization in sens mutants, and the increased dendritic branching as a result of Su(z)12(RNAi) or ab(RNAi) is epistatic to the increased dorsal extension and reduced lateral branching of sens mutants. In contrast, RNAi of the group A genes cg1244 and cg1841, which caused reduced arborization following RNAi in wild-type embryos, led to a reduction in primary dendrite outgrowth and lateral dendrite branching compared with injected controls. Therefore, at least in the instances described above, loss of group A genes is epistatic to loss of group B genes (Parrish, 2006).

A genetic screen in Drosophila for genes interacting with senseless during neuronal development identifies the importin moleskin

Senseless (Sens) is a conserved transcription factor required for normal development of the Drosophila peripheral nervous system. In the Drosophila retina, sens is necessary and sufficient for differentiation of R8 photoreceptors and interommatidial bristles (IOBs). When Sens is expressed in undifferentiated cells posterior to the morphogenetic furrow, ectopic IOBs are formed. This phenotype was used to identify new members of the sens pathway in a dominant modifier screen. Seven suppressor and three enhancer complementation groups were isolated. Three groups from the screen are the known genes Delta, lilliputian, and moleskin/DIM-7 (msk), while the remaining seven groups represent novel genes with previously undefined functions in neural development. The nuclear import gene msk was identified as a potent suppressor of the ectopic interommatidial bristle phenotype. In addition, msk mutant adult eyes are extremely disrupted with defects in multiple cell types. Reminiscent of the sens mutant phenotype, msk eyes demonstrate reductions in the number of R8 photoreceptors due to an R8 to R2,5 fate switch, providing genetic evidence that Msk is a component of the sens pathway. Interestingly, in msk tissue, the loss of R8 fate occurs earlier than with sens and suggests a previously unidentified stage of R8 development between atonal and sens (Pepple, 2007).

Sens, along with its homologs Gfi-1 and Pag-3, comprises a conserved family of proteins required for normal neural development. In Drosophila, sens is both necessary and sufficient for development of the PNS. In mice, loss of Gfi-1 leads to neurodegeneration of cerebellar Purkinje cells and sensoneural deafness due to loss of inner ear hair cells. Despite the obvious importance of the GPS proteins in normal neural development and their place near the top of the neuronal development cascade, few targets of these proteins in the process of neurogenesis are known. To identify members of this pathway required in neurogenesis, an F₁ dominant modifier screen was performed using an ectopic Sens phenotype in Drosophila. Advantage was taken of a dominant, modifiable phenotype generated by ectopic expression of Sens in undifferentiated cells posterior to the morphogenetic furrow. This ectopic Sens led to the recruitment of undifferentiated cells to the bristle fate (Pepple, 2007).

Both known and novel genes have been identified as potential members of the sens pathway by their ability to modify an ectopic Sens phenotype. The Notch signaling pathway is known to regulate Sens function during the resolution of the proneural cluster. This interaction was identified in the screen by the ability of heterozygous loss of Dl to enhance the ectopic Sens phenotype. The nuclear import gene moleskin (msk) was able to strongly suppress the effect of ectopic Sens. msk plays a role in normal eye development and R8 photoreceptor differentiation. Identification of the genes that are represented in the remaining complementation groups will lead to a better understanding of the GPS pathway and normal neural development. It is likely that the remaining complementation groups represent components of the Sens pathway due to their specific effect on lz and not the secondary screens as well as their requirement for normal bristle development in adult thoracic clones. Further characterization of these genes will offer new insight into the highly conserved Sens pathway (Pepple, 2007).

Alleles of msk were found to be suppressors of lz (the expression of UAS-sens in undifferentiated cells by the lozenge-GAL4 driver) with the highest frequency of any complementation group in the EMS screen. Usually such high representation of alleles indicates that the gene has an important role in the phenotype being tested and/or is readily mutagenized. The results presented here suggest a model in which Msk plays a role in the sens pathway. Initial observations of the effect of Msk on the lz phenotype suggested that Msk was needed to maintain high levels of Sens expression. It is possible that in this ectopic situation, Msk contributes to Sens import, but more likely Msk contributes to Sens expression indirectly by importing another component of the pathway that regulates Sens expression. Characterization of the ey-GAL4, UAS-flp (EGUF); msk phenotype strongly suggests that Msk is not the only import factor involved in the Sens pathway during normal development. Clearly, there is functional redundancy with another importin since complete loss of Msk function during early eye development does not remove Sens expression in all R8 cells. In third instar discs, Msk appears to play a role in the maintenance of the R8 cell fate very early in development. Little is known about the early stages of R8 differentiation after specification by Atonal. Previous work on R8 specification and development outlined a hierarchy of events in which Atonal is expressed first and appears to simultaneously activate expression of the downstream targets sens and sca-lacZ. Work on the sens phenotype determined that sca-lacZ expression is still present in sens clones, thereby establishing an epistatic relationship between sca-lacZ expression and sens. The data indicate that there is yet another step in the relationship between Atonal and these two downstream factors. The data suggest that in the msk eye, after specification of the R8 by Atonal but before the onset of sca-lacZ expression, R8 development is disrupted in some clusters, leading to an R2,5 fate switch. This is the first genetic evidence for factors positioned between ato and sca-LacZ/sens (Pepple, 2007).

Nuclear transport is required for the viability of all cells. Interestingly, the loss or decrease in function of some importins can cause specific defects during development. For example, the nuclear exportin Dcas is required for the export of Importin α3 in Drosophila. While null mutants in dcas are not viable, hypomorphs lead to specific cell fate changes in mechanosensory bristles. This phenotype is likely due to extreme sensitivity of Notch signaling to disruption of nuclear transport of one of its pathway members by Importin α3. It is possible that the Msk/Sens interaction was detectable for a similar reason. In the Sens gain-of-function situation, the high level of Sens required to generate ectopic bristles is very sensitive to decreased Msk levels, while during wild-type SOP differentiation, Sens is far less sensitive to Msk levels and exhibits only sporadic effects (Pepple, 2007).

One question still remains: How does the EGUF; msk eye survive at all given the important cargo that Msk is known to transport? The functional redundancy in the Importin family likely provides the cell with enough transport for survival and development in the absence of Msk. However, this idea raises a new question: Why was only Msk identified in the screen and no other importins? A model is proposed in which Msk is the key importin utilized by the cell for high levels of signaling. The lz phenotype requires high levels of signaling to generate ectopic bristles, and this model would explain why an effect with Msk and no other importin was detected. The model does not preclude the ability of other importins to provide transport redundancy for Msk cargos, and in fact evidence is seen for this redundancy in the ability of the EGUF; msk eye to survive and produce some normal ommatidia. Another importin must have the ability to import some level of Sens, pMAPK, and other unidentified factors into the nucleus. Data existst that indirectly support such a model for the role of Msk. In the Atonal intermediate groups within the morphogenetic furrow, Msk must be sequestered away from the nucleus to prevent the very high levels of cytoplasmic pMAPK from entering the nucleus. Although whether other nuclear importins are also sequestered to block pMAPK nuclear entry was not tested, overexpression of Msk in the intermediate groups allows pMAPK to enter the nucleus and affect nuclear signaling. The fact that the cell needs to sequester Msk to prevent high levels of EGFR pathway signaling supports a model in which Msk is important for high levels of signaling (Pepple, 2007).

It has been suggested in other developmental systems that importins are part of a mechanism that regulates the nuclear protein composition of transcription factors and chromatin remodeling factors. In Drosophila, Msk has been shown to import two other developmentally significant cargos, pMAPK and Caudal. In addition to these previously defined roles, the additional data that Msk and nucleocytoplasmic transport play an important role in Sens expression and R8 development. Perhaps more importantly, the fact that abnormalities seen in msk mutant eye discs arise between Atonal and Senseless expression suggests roles for as-yet undiscovered factors and new modes of regulation in this critical pathway (Pepple, 2007).

Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons

Specificity of sensory neurons requires restricted expression of one sensory receptor gene and the exclusion of all others within a given cell. In the Drosophila retina, functional identity of photoreceptors depends on light-sensitive Rhodopsins (Rhs). The much simpler larval eye (Bolwig organ; see The Extraretinal Eyelet of Drosophila: Development, Ultrastructure, and Putative Circadian Function) is composed of about 12 photoreceptors, eight of which are green-sensitive (Rh6) and four blue-sensitive (Rh5). The larval eye becomes the adult extraretinal 'eyelet' composed of four green-sensitive (Rh6) photoreceptors. This study shows that, during metamorphosis, all Rh6 photoreceptors die, whereas the Rh5 photoreceptors switch fate by turning off Rh5 and then turning on Rh6 expression. This switch occurs without apparent changes in the programme of transcription factors that specify larval photoreceptor subtypes. It was also shown that the transcription factor Senseless (Sens) mediates the very different cellular behaviours of Rh5 and Rh6 photoreceptors. Sens is restricted to Rh5 photoreceptors and must be excluded from Rh6 photoreceptors to allow them to die at metamorphosis. Finally, Ecdysone receptor (EcR) was shown to function autonomously both for the death of larval Rh6 photoreceptors and for the sensory switch of Rh5 photoreceptors to express Rh6. This fate switch of functioning, terminally differentiated neurons provides a novel, unexpected example of hard-wired sensory plasticity (Sprecher, 2008).

The adult Drosophila eyelet comprises approximately four photoreceptors located between the retina and the optic ganglia. It directly contacts the pacemaker neurons of the adult fly, the lateral neurons. In conjunction with the compound eye and the clock-neuron intrinsic blue-sensitive receptor cryptochrome it helps shift the phase of the molecular clock in response to light. All eyelet photoreceptors express green-sensitive Rh6, and are derived from photoreceptors of the larval eye that mediate light avoidance and entrainment of the molecular clock by innervating the larval lateral neurons (Sprecher, 2008).

Larval photoreceptors develop in a two-step process during embryogenesis. Primary precursors are specified first and develop as the four Rh5-subtype photoreceptors. They signal through Epidermal growth factor receptor (EGFR) to the surrounding tissue to develop as secondary precursors, which develop into the eight Rh6-subtype photoreceptors. Two transcription factors specify larval photoreceptor subtypes. Spalt (Sal) is exclusively expressed in Rh5 photoreceptors, where it is required for Rh5 expression. Seven-up (Svp) is restricted to Rh6 photoreceptors, where it represses sal and promotes Rh6 expression. A third transcription factor, Orthodenticle (Otd), expressed in all larval photoreceptors, acts only in the Rh5 subtype to promote Rh5 expression and to repress Rh6 (Sprecher, 2008 and references therein).

To address the relation between the larval Rh5 and Rh6 photoreceptors and the adult eyelet, they were tracked through metamorphosis. To permanently label them, UAS-Histone2B::YFP, which is stably incorporated in the chromatin, and thus remains detectable in post-mitotic neurons throughout pupation, was used. Surprisingly, all Rh6 photoreceptors degenerate and disappear during early phases of metamorphosis. In contrast, Rh5 photoreceptors can be followed throughout pupation. Expression of Rh5 ceases during early stages of pupation and, at mid-pupation, neither Rh5 nor Rh6 can be detected. About four cells are still present, however, and can be identified by rh5-Gal4/UAS-H2B::YFP or GMR-Gal4/UAS-H2B::YFP. Eyelet photoreceptors only express Rh6, even though H2B::YFP driven by rh5-Gal4 is detectable in those cells. Therefore, the four larval Rh5 photoreceptors must switch rhodopsin expression at metamorphosis to give rise to the four eyelet Rh6 photoreceptors. The remaining eight Rh6 photoreceptors die, their axon becoming fragmented before disappearing. A 'memory experiment' (rh5-Gal4/UAS-Flp;Act-FRT > STOP > FRT-nlacZ) also showed that eyelet Rh6 photoreceptors did express Rh5 earlier (Sprecher, 2008).

The death of Rh6 photoreceptors and transformation of Rh5 photoreceptors was further verified by three independent sets of experiments (Sprecher, 2008).

(1) Rh5 photoreceptors were ablated by expressing pro-apoptotic genes rpr and hid (rh5-Gal4/UAS-rpr,UAS-hid). This results in the absence of larval Rh5 photoreceptors and the complete absence of the eyelet. Conversely, preventing cell death of the Rh6 subtype by expressing the apoptosis inhibitor p35 (rh6-Gal4/UAS-p35) leads to an eyelet that consists of 12 photoreceptors, all expressing Rh6 (Sprecher, 2008).

(2) Larval Rh6 photoreceptors development was blocked by expressing a dominant negative form of EGFR (so-Gal4/UAS-H2B::YFP; UAS-EGFR^DN). The eyelet of these animals is not affected and three or four cells express Rh6 normally. This shows that larval Rh6 photoreceptors do not contribute to the eyelet (Sprecher, 2008).

(3) The expression of Sal (Rh5-subtype specific) and Svp (Rh6-subtype specific) was analyzed in the adult eyelet: eyelet photoreceptors still express Sal, but not Svp even though these photoreceptors now express Rh6. Rh5 requires Sal expression in the Bolwig organ, but Otd function is also necessary to activate Rh5 and to repress Rh6. In otd mutants, larval Rh5 photoreceptors marked by Sal express Rh6 and lack Rh5 expression, thus mimicking the switch at metamorphosis. Thus, Rh6 could be expressed in Rh5 photoreceptors if otd function were lost in the eyelet. However, Otd expression does not change during the transition from the Bolwig organ to eyelet although it might be inactive in the eyelet (Sprecher, 2008).

What is the trigger that controls the switch from rh5 to rh6? Ecdysone controls many developmental processes during metamorphosis. EcR is expressed during the third larval instar and pupation in all larval photoreceptors and surrounding tissues. To evaluate EcR activity, a reporter line was used in which lacZ is under the control of multimerized ecdysone response elements (7XEcRE-lacZ). The expression of lacZ is absent until late third instar and prepupation, whereas thereafter all larval photoreceptors (and surrounding tissue) express 7XEcRE-lacZ. EcR expression decreases during late pupation and is no longer detectable by the time Rh6 expression starts in the eyelet (Sprecher, 2008).

To test the role of ecdysone, a dominant negative form of EcR was expressed specifically in larval Rh5 photoreceptors, while permanently labelling these cells (rh5-Gal4/UAS-H2B::YFP;UAS-EcR^DN). This causes no disruption of larval photoreceptor fate, but the eyelet of these animals now consists of four photoreceptors that all express Rh5 instead of Rh6. A comparable phenotype is observed after expression of an RNA interference (RNAi) construct for EcR (rh5-Gal4/UAS-H2B::YFP;UAS-EcR^RNAi). Therefore, loss of EcR function prevents larval photoreceptors from switching to Rh6 expression. In both cases, larval Rh6 photoreceptors still degenerate and are not observed in the eyelet (Sprecher, 2008).

The dominant negative form of EcR was also expressed in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP; UAS-EcR^DN). In this case, the Bolwig organ is not affected but the resulting adult eyelet consists of about 12 photoreceptors, all expressing Rh6. This presumably results from Rh6 photoreceptors not undergoing apoptosis whereas larval Rh5 photoreceptors still switch expression to Rh6 in the eyelet. Expression of UAS-EcR-RNAi in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-EcR^RNAi) leads to the same results (Sprecher, 2008).

Although EcR could directly control the switch of rhodopsin expression through binding to the promoters of rh5 and rh6, these promoters contain no potential EcR binding sites. Moreover, as no EcR expression is detectable when Rh6 starts to be expressed, this would make it unlikely for EcR to control directly the switch to Rh6. Finally, only allowing expression of the dominant negative form of EcR starting at mid-pupation (GMR-Gal4/Tub-Gal80^ts,UAS-EcR^DN), after rh5 is switched off, does not prevent activation of Rh6 in the eyelet. Thus EcR most likely acts in an indirect manner in regulating rhodopsins, likely through the activation of transcription factors that bind to rh5 and rh6 promoters (Sprecher, 2008).

The differential response to ecdysone of Rh6 photoreceptors (which die) and of Rh5 photoreceptors (which switch to Rh6) must be due to intrinsic differences between the two subtypes before EcR signalling. Likely candidates are Sal and Svp. However, late misexpression of Svp in Rh5 photoreceptors (rh5-Gal4/UAS-H2B::YFP;UAS-svp) or of Sal in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-sal) neither affects rhodopsin expression or cell number in the eyelet nor alter the expression of rhodopsins in the Bolwig organ (which is only affected by very early expression of these transcription factors, through so-Gal4. Thus neither Sal nor Svp are sufficient to alter the response of larval photoreceptors to EcR (Sprecher, 2008).

An additional factor, independent from svp and sal, must therefore allow survival of Rh5 photoreceptors, or promote Rh6 photoreceptor death. It was found that the transcription factor Sens is specifically expressed in larval Rh5 photoreceptors and remains expressed in all cells in the eyelet where it might act to promote cell survival. To test this, sens was misexpressed in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-sens). This results in an eyelet that consists of 12 photoreceptors, all expressing Rh6. Thus, expression of Sens in Rh6 photoreceptors is sufficient to rescue them from death, without affecting Sal and Svp expression and subtype specification of larval photoreceptors (Sprecher, 2008).

Ecdysone hormonal signalling thus acts in two independent ways during the formation of the adult eyelet. First, it induces the degeneration of the Rh6 subtype, thereby assuring the correct number of eyelet photoreceptors. This apoptotic death requires the absence of Sens, whose expression is restricted to Rh5 photoreceptors that survive. Second, ecdysone signalling is also required to trigger the switch of spectral sensitivity of blue-sensitive (Rh5) larval photoreceptors to green-sensitive (Rh6) eyelet photoreceptors (Sprecher, 2008).

Thus terminally differentiated sensory neurons switch specificity by turning off one Rhodopsin and replacing it with another. Although examples of such switches in sensory specificity of terminally differentiated, functional, sensory receptors are extremely rare, this strategy might be more common than currently anticipated. In the Pacific pink salmon and rainbow trout, newly hatched fish express an ultraviolet opsin that changes to a blue opsin as the fish ages. As in flies, this switch might reflect an adaptation of vision to the changing lifestyle. The maturing salmon, born in shallow water, later migrates deeper in the ocean where ultraviolet does not penetrate. The rhodopsin switch in the eyelet may similarly be an adaptation to the deeper location of the eyelet within the head, as light with longer wavelengths (detected by Rh6) penetrates deeper into tissue than light with shorter wavelengths (detected by Rh5) (Sprecher, 2008).

The eyelet functions with retinal photoreceptors and Cryptochrome to entrain the molecular clock in response to light. The larval eye, on the other hand, functions in two distinct processes: for the entrainment of the clock and for the larva to avoid light. Interestingly, the Rh5 subtype appears to support both functions whereas Rh6 photoreceptors only contribute to clock entrainment. Thus, the photoreceptor subtype that supports both functions of the larval eye is the one that is maintained into the adult and becomes the eyelet. Why are Rh6-sensitive photoreceptors not maintained? As these photoreceptors are recruited to the larval eye secondarily, the ancestral Bolwig organ might have had only Rh5 photoreceptors and had to undergo a switch in specificity. Larval Rh5 photoreceptors appear to maintain their overall connectivity to the central pacemaker neurons. However, they are also profoundly restructured and exhibit widely increased connectivity during metamorphosis. This might be due to the increase in number of their target neurons, and the switch of Rh might be part of more extensive plasticity during formation of the eyelet, including increased connectivity and possibly the innervation of novel target neurons (Sprecher, 2008).

The general model that sensory neurons express only a single sensory receptor gene does not hold true for salmon and the fruitfly. Interestingly, reports from several other species, including amphibians, rodents and humans, show co-expression of opsins. In humans, for instance, it has been proposed that cones first express S opsin and later switch to L/M opsin. However, this likely reflects a developmental process rather than a functional adaptation (Sprecher, 2008).

This study identified two major players in the genetic programme for the transformation of the larval eye to the eyelet. (1) EcR acts as a trigger for both rhodopsin switch and apoptosis. Surprisingly, the upstream regulators specifying larval photoreceptor-subtype identity, Sal, Svp and Otd, do not contribute to the genetic programme of sensory plasticity of the rhodopsin switch. Therefore a novel genetic programme is required for regulating rhodopsin expression in the eyelet, which likely depends on downstream effectors of EcR (Sprecher, 2008).

(2) Larval Rh5 and Rh6 photoreceptors respond differently to ecdysone, either switching rhodopsin expression or undergoing apoptosis. This appears to depend on Sens, which is likely to be required for the survival of Rh5 photoreceptors. The role of Sens in inhibiting apoptosis is not unique to this situation: Sens is essential to promote survival of salivary-gland precursors during embryogenesis. The vertebrate homologue of sens, Gfi-1, acts to inhibit apoptosis of T-cell precursors in haematopoiesis and cochlear hair cells of the inner ear. Thus the anti-apoptotic function of Sens/Gfi-1 may be a general property of this molecule (Sprecher, 2008).

Ecdysone acts in remodelling neurons during metamorphosis. In γ-neurons of the mushroom body, a structure involved in learning and memory, ecdysone is required for the pruning of larval processes. Similarly, dendrites of C4da sensory neurons undergo large-scale remodelling that depends on ecdysone signalling. Interestingly, in the moth Manduca, 'lateral neurosecretory cells' express cardio-acceleratory peptide 2, which is switched off in response to ecdysone before expression of the neuropeptide bursicon is initiated in the adult (Sprecher, 2008).

The transformation of larval blue-sensitive photoreceptors to green-sensitive photoreceptors of the eyelet reveals an unexpected example of sensory plasticity by switching rhodopsin gene expression in functional, terminally differentiated sensory neurons (Sprecher, 2008).

The enhancer of trithorax and polycomb gene Caf1/p55 is essential for cell survival and patterning in Drosophila development

In vitro data suggest that the human RbAp46 and RbAp48 genes encode proteins involved in multiple chromatin remodeling complexes and are likely to play important roles in development and tumor suppression. However, to date, understanding of the role of RbAp46/RbAp48 and its homologs in metazoan development and disease has been hampered by a lack of insect and mammalian mutant models, as well as redundancy due to multiple orthologs in most organisms studied. This study reports the first mutations in the single Drosophila RbAp46/RbAp48 homolog Caf1, identified as strong suppressors of a senseless overexpression phenotype. Reduced levels of Caf1 expression result in flies with phenotypes reminiscent of Hox gene misregulation. Additionally, analysis of Caf1 mutant tissue suggests that Caf1 plays important roles in cell survival and segment identity, and loss of Caf1 is associated with a reduction in the Polycomb Repressive Complex 2 (PRC2)-specific histone methylation mark H3K27me3. Taken together, these results suggest suppression of senseless overexpression by mutations in Caf1 is mediated by participation of Caf1 in PRC2-mediated silencing. More importantly, the mutant phenotypes confirm that Caf1-mediated silencing is vital to Drosophila development. These studies underscore the importance of Caf1 and its mammalian homologs in development and disease (Anderson, 2011).

Several lines of evidence suggest that the participation of Caf1 in PcG complexes may account for many of the phenotypes observed in flies with altered expression of Caf1. First, Caf1 loss-of-function clones in the eye have phenotypes ranging from slight disorganization and bristle defects to almost complete loss of homozygous tissue in adults, and incomplete rescue of Caf1 results in adult eyes that are small and disorganized. Clones of many PcG genes have similar phenotypes in the eye. Loss of E(z) or Pc causes mild defects in differentiation in the third instar disc, but clones fail to survive in adults. An analogous situation occurs in Caf1^short clones, where expression of Elav, which marks differentiating neurons, is present in Caf1 clones at third instar but Caf1^short tissue is largely missing in the adult. Derepression of Hox genes could account for these phenotypes, as ectopic expression of many Hox genes in the eye field causes small disorganized eyes in adults (Anderson, 2011).

Second, flies with incomplete rescue of Caf1 display a range of homeotic phenotypes, notably transformation of arista to leg. Similar homeotic transformations, including antenna-to-leg transformations, are a hallmark of mutations in PcG genes. Also a genetic interaction was observe between Caf1 and the PRC1 gene Pc, since mutations in Caf1 are able to dominantly suppress the homeotic transformation of second or third leg to first leg in Pc¹⁵/+ males (Anderson, 2011).

Third, the disrupted patterning of Caf1^short mutant heads may also result from PcG dysfunction. It is possible that these patterning defects are non-cell autonomous and may be an indirect result of widespread apoptosis in the eye disc. Normally, when an imaginal disc is injured, remaining cells proliferate and assume correct identities, leading to a perfectly patterned adult structure. This type of regeneration requires that some determined cells must change their fates and involves substantial chromatin remodeling. Under specific circumstances, the disc can regenerate with incorrect patterning, leading to duplication, deletion or transformation of structures, a phenomenon referred to as transdetermination. Levels of many PcG transcripts are increased in transdetermining imaginal discs, and heterozygous mutations in PcG genes can enhance transdetermination in regenerating imaginal discs. Therefore, one interpretation of the patterning defects in Caf1^short mutant discs is that under the stress of widespread apoptosis, the remaining heterozygous tissue is haploinsufficient for the chromatin remodeling activity required to properly regenerate and pattern the injured disc. Consistent with this interpretation, no extra or missing appendages are observed in flies with Caf1^long clones, which show less active Caspase 3 staining at third instar (Anderson, 2011).

Finally, in Caf1 mutant tissue, a reduction is observed in levels of the H3K27me3 mark, which is associated with inactive chromatin and PRC2 activity. These data are consistent with a disruption of PRC2 function as a result of loss of Caf1 and represent the first in vivo evidence that Caf1 is an essential member of this chromatin remodeling complex in an animal model (Anderson, 2011).

One obvious question arises from the current study: why were multiple Caf1 alleles identified in a screen for modifiers of the sens overexpression phenotype? Moreover, it is surprising that mutations in Caf1 were not identified in previous Drosophila modifier screens involving PcG or Rb pathway members. It is proposed that the link between sens and Caf1 is due to the role of Caf1 in PcG-mediated silencing (Anderson, 2011).

Recent evidence suggests that Sens and Hox proteins can compete for binding at overlapping sites at an enhancer of the rhomboid (rho) locus (Li-Kroeger, 2008). When the Hox protein Abdominal-A (Abd-A) binds, transcription of rho is activated, whereas binding by Sens leads to repression of rho. In the embryo, this mechanism acts as a molecular switch to allow differentiation of either chordotonal organs (under control of Sens) or hepatocyte-like cells called oenocytes (by the action of Abd-A). It is proposed that a similar mechanism underlies the suppression of the Sens overexpression phenotype. It is hypothesized that one or more targets of Sens in the eye contain similar overlapping sites that can be bound by either Sens or a Hox protein. During normal development, these loci are bound by neither Sens nor Hox in undifferentiated cells posterior to the furrow, as no Hox genes are known to be widely expressed in the eye field. In the absence of both types of factors, these loci are transcriptionally active, and are necessary to ultimately attain the proper fates of the cells in which they are expressed. When Sens is overexpressed, as in ls, Sens binds to its recognition site in the downstream loci, repressing transcription. Repression of these genes initiates a cascade leading to a change in cell fate; for example, some of the cells that would normally become secondary or tertiary pigment cells now become bristle precursors, giving rise to the extra bristles of ls. However, when one copy of Caf1 is lost, a slight derepression of the Hox genes occurs due to loss of PcG activity. Hox proteins are now able to compete with Sens for the overlapping binding sites, tipping the balance towards activation of downstream genes and attainment of normal cell fate - effectively suppressing ls. The ability of ectopic expression of pb and Antp in the eye to suppress ls is consistent with this hypothesis. Suppression of ls by Hox proteins is particularly significant given that ectopic expression of Antp alone in the eye field leads to a small and disorganized eye. As Sens activity is exquisitely sensitive to Hox proteins, especially in the eye, the screen for modifiers of a sens overexpression phenotype was therefore ideal for identifying mutations in Caf1 (Anderson, 2011).

Previous studies have explored pro-apoptotic roles of Hox proteins and anti-apoptotic roles of Sens. It is therefore possible that one effect of Hox gene derepression in ls eyes suppressed by Caf1 may be restoration of an apoptotic fate in cells that would otherwise form bristle precursors due to ectopic Sens. Abd-A expression in the abdomen during normal third instar larval development leads to apoptosis of proliferating neuroblasts of the central nervous system, and ectopic expression of other Hox genes can also cause neuroblast apoptosis. Accordingly, survival of neuroblasts is dependent on PcG activity to repress Hox gene expression. Furthermore, expression of Sens is necessary in the Drosophila embryonic salivary gland to prevent apoptosis. Thus, one possible mechanism for suppression of ls by Caf1 mutations is that in the ls eye, Sens may promote the ectopic bristle fate partly by repressing apoptotic genes in cells normally fated to die, whereas in the ls eye suppressed by mutations in Caf1, ectopic Hox proteins may promote apoptosis and prevent bristle formation. Increased Ubx expression was not detected by antibody staining; however, a very small increase in one or more Hox proteins may be all that is necessary to change the transcriptional state of downstream loci and prevent the ectopic bristles and other defects in the highly sensitized ls eye - especially in the eye field, where no Hox genes are known to be highly expressed. Furthermore, the fact that multiple Hox proteins can recognize the same DNA binding site offers the possibility that the competitive effect of each Hox protein type on genes with overlapping Sens/Hox binding sites would be additive. Therefore, although loss of one copy of Caf1 may only cause a small derepression of any one Hox gene, mild derepression of many Hox genes collectively can lead to strong repression of the ls phenotype (Anderson, 2011).

Biochemical evidence suggests that Caf1 is a member of multiple complexes that effect gene regulation through chromatin remodeling, suggesting that it is a vital component of the cell's arsenal of chromatin modifying factors. Although the results suggest that disruption of PRC2 function may be the most important consequence of Caf1 gain- or loss-of-function, many phenotypes that were observed in Caf1 mutant tissue are also reminiscent of mutations in members of other complexes previously shown to contain Caf1. It is not surprising that all three alleles of Caf1 in the current study are homozygous lethal, and that Caf1^short cells have poor viability, considering that Caf1 has been found in the NURF and CAF-1 complexes, which have fundamental roles in nucleosome assembly and spacing. The apoptosis observed in eyes with Caf1^short clones is also consistent with a role for Caf1 in the dREAM (Drosophila Rbf, E2F2, and Myb-interacting proteins) complex. Members of the E2f family of transcription factors can complex with Dp proteins and bind short recognition sites to activate transcription. When Rb binds the E2f-Dp complex, transcription is repressed. Like Caf1 homozygotes, homozygous rbf1 null flies die in early larval development. Fully rbf1-deficient embryos display increased apoptosis, a phenotype reminiscent of the increased active Caspase-3 staining seen anterior to the morphogenetic furrow in eye discs with Caf1^short clones (Anderson, 2011).

The mammalian homologs of sens, Growth Factor Independence 1 (Gfi1) and Gfi1b are essential to the development of multiple cell types and have been implicated as oncogenes. Therefore, the possibility that Caf1 links Sens with the activity of PcG complexes through parallel, competing pathways has implications for both Drosophila development and the activity of Gfi1 family members in human development and disease, and warrants additional study beyond the scope of the present work. The results underscore the importance of Caf1 to diverse processes, including cell survival and tissue identity, and highlight the participation of Caf1 in multiple chromatin remodeling complexes. Further studies are needed to fully assess the importance of Caf1 in Drosophila development, as well as its developmental role in other chromatin remodeling complexes (Anderson, 2011).

EVOLUTIONARY HOMOLOGS

pag-3 - a Lyra homolog in C. elegans

Mutations in a newly identified gene, pag-3, cause ectopic expression of touch neuron genes meo-7, mec-7lacZ and mec-4lacZ in the lineal sisters of the ALM touch neurons, the BDU neurons. pag-3 mutants also show a reverse kinker uncoordinated phenotype. The first pag-3 allele was isolated in a screen for mutants with altered immunofluorescence staining patterns. Two additional pag-3 alleles have been identified in a noncomplementation screen of 38,000 haploid genomes. All of the pag-3 alleles are recessive to wild type and cause the same phenotypes. Two-factor crosses, deficiency mapping and three-factor crosses locate pag-3 to the right arm of the X chromosome between unc-3 and unc-7. Because recessive mutations in pag-3 result in expression of several touch cell specific genes in the BDU neurons, wild-type pag-3 must directly or indirectly suppress expression of these genes in the BDU neurons. Although pag-3 mutants do not show mec-3lacZ expression in their BDU neurons, expression of mec-7lacZ and mec-4lacZ in the BDU neurons of pag-3 mutants requires mec-3 (Jia, 1996).

Mutations in the C. elegans gene pag-3 result in misexpression of touch receptor-specific genes in the BDU interneurons and in motility defects. pag-3 encodes a C2H2-type zinc finger protein related to the mammalian GFI-1 protein. Sequencing of the three pag-3 alleles shows that two apparent null alleles encode a nonsense mutation before the zinc fingers and a missense mutation in the fourth zinc finger that changes a coordinating histidine to a tyrosine. The third allele contains a nonsense mutation in the N-terminal region but is not a null allele. Northern analysis shows that a single pag-3 transcript of about 1.6 kb is present in embryos and L1, L2 and L3 larvae. pag-3 message levels are about twofold higher in pag-3 mutants than in wild-type animals, which suggests that pag-3 may negatively regulate its own expression. pag-3lacZ fusion genes are expressed in the BDU interneurons, the touch neurons, 11 VA and 11 VB ventral cord motor neurons, two AVF interneurons and in unidentified neurons of the retrovesicular ganglion. The BDU neurons and the ALM touch neurons are lineal sister cells in the AB.a lineage and the VA and VB motor neurons are lineal sister cells in the AB.p lineage. The VA motor neurons are required for backward movement and the VB motor neurons are required for forward movement. Mosaic analysis has shown that the wild-type pag-3 gene is required in the AB.p lineage for coordinated movement and in the AB.a lineage to suppress touch neuron gene expression in the BDU neurons. Because pag-3 is expressed in both the BDU neurons and in the touch neurons, another protein(s) not expressed in the touch neurons may interact with pag-3 to repress touch neuron gene expression in the BDU neurons. Alternatively, another protein in the touch receptor cells may inactivate PAG-3 and allow expression of the touch receptor program. These results show that pag-3 is a temporally regulated gene that is expressed early in development and functions in multiple types of neurons. They also strongly suggest that the PAG3 protein is a DNA-binding protein with properties similar to the mammalian proto-oncogene product GFI-1 (Jia, 1997).

The Caenorhabditis briggsae homolog of the C. elegans pag-3 gene was cloned and sequenced. When transformed into a C. elegans pag-3 mutant, the C. briggsae pag-3 gene rescues the pag-3 reverse kinker and lethargic phenotypes. The C. elegans pag-3 gene fused to lacZ is expressed in the same pattern in C. elegans and C. briggsae. Unlike many gene homologs compared between C. elegans and C. briggsae, extensive sequence conservation is found in the non-coding regions upstream of the pag-3 exons, in several of the introns and in the downstream non-coding region. Furthermore, the splice acceptor and splice donor sites are conserved, and the size of the introns and exons is surprisingly similar. The predicted protein sequence of C. briggsae PAG-3 is 85% identical to the protein sequence of C. elegans PAG-3. Because so much of the non-coding region of pag-3 is conserved, the control of pag-3 may be quite complex, involving the binding of many trans-acting factors. These results suggest the evolutionary conservation of the pag-3 gene sequence, its expression and function (Aamodt, 2000).

During C. elegans development, the patterns of cell divisions, cell fates and programmed cell deaths are reproducible from animal to animal. In a search for mutants with abnormal patterns of programmed cell deaths in the ventral nerve cord, mutations were identified in the gene pag-3, which encodes a zinc-finger transcription factor similar to the mammalian Gfi-1 and Drosophila Senseless proteins. In pag-3 mutants, specific neuroblasts express the pattern of divisions normally associated with their mother cells, producing with each reiteration an abnormal anterior daughter neuroblast and an extra posterior daughter cell that either terminally differentiates or undergoes programmed cell death, which accounts for the extra cell corpses seen in pag-3 mutants. In addition, some neurons do not adopt their normal fates in pag-3 mutants. The phenotype of pag-3 mutants and the expression pattern of the PAG-3 protein suggest that in some lineages pag-3 couples the determination of neuroblast cell fate to subsequent neuronal differentiation. It is proposed that pag-3 counterparts in other organisms determine blast cell identity and for this reason may lead to cell lineage defects and cell proliferation when mutated (Cameron, 2002).

The PVQ and BDU neurons in pag-3 mutants are abnormal, despite being generated by apparently normal cell lineages, suggesting that these neurons fail to differentiate properly in pag-3 mutants. The VA motoneurons also differentiate abnormally in pag-3 mutants, as demonstrated by the defective backwards movement of these animals and by abnormal expression of an unc-4 reporter gene. A role for pag-3 in differentiating and differentiated neurons is supported by the expression pattern of the protein. In the Q neuroblast lineages PAG-3 protein is detected only after the generation of the terminally differentiating AVM and PVM mechanosensory neurons. In many neurons, including the PVQ, BDU and touch neurons, PAG-3 protein is present throughout the life of the animal. Some of these cells are abnormal in pag-3 mutants (the BDU and PVQ neurons), while others have no obvious defect. These data suggest that pag-3 functions in diverse contexts within the developing nervous system, with an essential role in the development of some neuroblasts and neurons and a currently unapparent role in other neurons (Cameron, 2002).

PAG-3 does not appear to be associated with any obvious single characteristic of differentiated neurons. In the ventral nerve cord, PAG-3 is expressed in cells that live and in cells that undergo programmed cell death; in cells that express lin-11 and in cells that do not; and transiently in the VA2-10 motoneurons but continuously in VA11 and VA12. PAG-3 is required for cell-fate determination by the Pn.aa neuroblasts but not by the Pn.ap neuroblasts. PAG-3 was expressed in adult animals in neurons of several different types, including motoneurons (VA11, VA12), sensory neurons (ALM, PLM, AVM and PVM) and interneurons (BDU, RIG and AVF). Given its extensive similarity to mammalian Gfi-1 transcription factors, PAG-3 most probably affects cell fates by regulating transcription. Rather than affecting expression of a single common set of genes in all neuronal lineages, pag-3 is expressed in many neuronal subtypes at different points in neuronal development suggesting that pag-3 cooperates with other factors to regulate the expression of cell type- and developmental stage-specific sets of genes to generate the complex pattern of neuronal subtypes seen in C. elegans (Cameron, 2002).

pag-3 expression is specifically activated in the Pn.aa neuroblasts, the descendants of which are abnormal in pag-3 mutants. By contrast, expression is not activated to a detectable level in the sisters of these cells, the Pn.ap neuroblasts, the descendants of which appeared normal in pag-3 mutants. These observations suggest that pag-3 acts specifically in the Pn.aa neuroblasts and their descendants. PAG-3 protein was present at the time of generation of neurons descended from each Pn.aa cell. It is shortly after the generation of the terminal cells in these lineages that these neurons begin to adopt identifying characteristics, such as class-specific patterns of axonal projections or the morphological characteristics of a dying cell. PAG-3 thus may well be expressed in response to Pn.aa lineage-specific signals to determine blast cell fates and then act later to induce distinctive differentiated features characteristic of the neurons produced by those lineages (Cameron, 2002).

The function of PAG-3 may be similar to that of UNC-86 (Drosophila homolog: Acj6), a POU-homeodomain protein that couples cell lineage cues to aspects of terminal differentiation. Like mutations in pag-3, mutations in unc-86 result in the reiteration of some neuroblast lineages. unc-86 is the only other C. elegans gene known to be able to mutate to cause reiterative cell lineage defects that specifically affect development of the nervous system. In addition to being required for neuroblast determination, unc-86 also specifies characteristics of the mechanosensory neurons generated by those neuroblasts. pag-3 may function similarly in the ventral nerve cord lineages. In these lineages, pag-3 determines Pn.aa neuroblast fate and may also establish the fate of the VA and VB motoneurons generated by those neuroblasts (Cameron, 2002).

Wnt/β-catenin signaling integrates patterning and metabolism of the insect growth zone

Wnt/β-catenin and Hedgehog (Hh) signaling are essential for transmitting signals across cell membranes in animal embryos. Early patterning of the principal insect model, Drosophila melanogaster, occurs in the syncytial blastoderm, where diffusion of transcription factors obviates the need for signaling pathways. However, in the cellularized growth zone of typical short germ insect embryos, signaling pathways are predicted to play a more fundamental role. Indeed, the Wnt/β-catenin pathway is required for posterior elongation in most arthropods, although which target genes are activated in this context remains elusive. This study used the short germ beetle Tribolium castaneum to investigate two Wnt and Hh signaling centers located in the head anlagen and in the growth zone of early embryos. Wnt/β-catenin signaling was found to act upstream of Hh in the growth zone, whereas the opposite interaction occurs in the head. The target gene sets of the Wnt/β-catenin and Hh pathways were determined; the growth zone signaling center activates a much greater number of genes and the Wnt and Hh target gene sets are essentially non-overlapping. The Wnt pathway activates key genes of all three germ layers, including pair-rule genes, and Tc-caudal (see Drosophila caudal) and Tc-twist (see Drosophila twist). Furthermore, the Wnt pathway is required for hindgut development and Tc-senseless (Drosophila Lyra/Senseless) as a novel hindgut patterning gene required in the early growth zone. At the same time, Wnt acts on growth zone metabolism and cell division, thereby integrating growth with patterning. Posterior Hh signaling activates several genes potentially involved in a proteinase cascade of unknown function (Oberhofer, 2014).

Gfi-1 homologs in fish and frogs

X-MyT1 is a C2HC-type zinc finger protein involved in the primary selection of neuronal precursor cells in Xenopus. Expression of this gene is positively regulated by the bHLH protein X-NGNR-1 and negatively regulated by the Notch/Delta signal transduction pathway. X-MyT1 is able to promote ectopic neuronal differentiation and to confer insensitivity to lateral inhibition, but only in cooperation with bHLH transcription factors. Inhibition of X-MyT1 function inhibits normal neurogenesis as well as ectopic neurogenesis caused by overexpression of X-NGNR-1. On the basis of these findings, it is suggested that X-MyT1 is a novel, essential element in the cascade of events that allows cells to escape lateral inhibition and to enter the pathway that leads to terminal neuronal differentiation (Bellefroid, 1996).

Zebrafish growth factor independent 1 (gfi1) expression is detected in the ganglion cells of the neural retina and in developing hair cells of the ear. In keeping with a role in the development of sensory hair cells, gfi1 is also expressed in neuromasts of the anterior and posterior lateral line system. Finally, gfi1 is expressed in the developing epithalamus in the dorsal diencephalon where its transcription is restricted to the parapineal (Dufourcq, 2004).

Gfi-1B (growth factor independence-1B) gene is an erythroid-specific transcription factor, whose expression plays an essential role in erythropoiesis. The human Gfi-1B promoter region has been defined; GATA-1 mediates erythroid-specific Gfi-1B transcription. By further investigating the regulation of the Gfi-1B promoter, this study reports that (1) Gfi-1B transcription is negatively regulated by its own gene product, (2) GATA-1, instead of Gfi-1B, binds directly to the Gfi-1-like sites in the Gfi-1B promoter and (3) Gfi-1B suppresses GATA-1-mediated stimulation of Gfi-1B promoter through their protein interaction. These results not only demonstrate that interaction of GATA-1 and Gfi-1B participates in a feedback regulatory pathway in controlling the expression of the Gfi-1B gene, but also provide the first evidence that Gfi-1B can exert its repression function by acting on GATA-1-mediated transcription without direct binding to the Gfi-1 site of the target genes. Based on these data, it is proposed that this negative auto-regulatory feedback loop is important in restricting the expression level of Gfi-1B, thus optimizing its function in erythroid cells (Huang, 2005).

Gfi-1 - a Lyra homolog in mammals

The proliferation of cultured rat Nb2 lymphoma cells is dependent on prolactin (PRL) acting as the principal growth factor. Previously, PRL-independent Nb2 sublines were obtained by PRL starvation of the parent line and cloning of surviving cells. Development of PRL independence has been, in some cases, associated with a reciprocal translocation involving chromosome 14 at breakpoint 14p22. A novel 14p22 zinc finger protein-encoding gene, Gfi-1, has been examined for a role in Nb2 cell proliferation. PRL-dependent Nb2 cells express the gene during active growth; in comparison, in stationary, early G1-arrested cells obtained by an 18 hr lactogen starvation, Gfi-1 gene expression is markedly decreased. Addition of PRL to such stationary cells leads to induction of Gfi-1 gene expression within a few hr with a maximum in late G1. Actively growing cells from 5 different PRL-independent Nb2 sublines, cultured in chemically defined, mitogen-free medium, express the gene constitutively. In two sublines, carrying the 14p22 rearrangement, the gene is markedly overexpressed. The results suggest the Gfi-1 gene product has a regulatory role in Nb2 cell mitogenesis and that unscheduled activation could contribute to loss of PRL dependency (Gilks, 1995).

The Gfi-1 proto-oncogene encodes a zinc finger protein with six C2H2-type, C-terminal zinc finger motifs and is activated by provirus integration in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence-specific manner. The Gfi-1 binding site, TAAATCAC(A/T)GCA, has been defined via random oligonucleotide selection utilizing a bacterially expressed glutathione S-transferase-Gfi-1 fusion protein. Binding to this site has been confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oliginucleotides have defined the relative importance of specific bases at the consensus binding site. Deletion of individual zinc fingers demonstrates that only zinc fingers 3, 4, and 5 are required for sequence-specific DNA binding. Potential Gfi-1 binding sites are detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early-chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3 fibroblasts, are repressed by Gfi-1, and the repression is abrogated by mutation of critical residues in the two Gfi-1 binding sites. These results suggest that Gfi-1 may play a role in HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes (Zweidler-Mckay, 1996).

The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a 20-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in IL-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by IL-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by IL-2 starvation, it is concluded that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation (Grimes, 1996a).

The rat and mouse Growth Factor Independence (Gfi-1) genes allow cells in culture to overcome the depletion of growth factors in the culture medium and maintain their proliferative potential. As part of a cloning strategy to isolated genes from human chromosome 1p22 that are associated with a constitutional chromosome translocation from a patient with stage 4S neuroblastoma, the human homolog of the Gfi gene has been identified and a 50 Kb map position has been identified within a well characterised YAC contig from the region. The full length cDNA sequence is 81% homologous with the rodent counterparts and, at the protein level, is even more highly conserved (Roberts, 1997).

After rearrangement of the T-cell receptor (TCR) ßlocus, early CD4(-)/CD8(-) double negative (DN) thymic T-cells undergo a process termed 'ßselection' that allows the preferential expansion of cells with a functional TCR ßchain. This process leads to the formation of a rapidly cycling subset of DN cells that subsequently develop into CD4(+)/CD8(+) double positive (DP) cells. Using transgenic mice that constitutively express the zinc finger protein Gfi-1 and the serine/threonine kinase Pim-1, it was found that the levels of both proteins are important for the correct development of DP cells from DN precursors at the stage where 'ßselection' occurs. Analysis of the CD25(+)/CD44(-,lo) DN subpopulation from these animals reveals that Gfi-1 inhibits and Pim-1 promotes the development of larger ßselected cycling cells ('L subset') from smaller resting cells ('E subset') within this subpopulation. It is concluded that both proteins, Pim-1 and Gfi-1, participate in the regulation of ßselection-associated pre-T-cell differentiation in opposite directions and that the ratio of both proteins is important for pre-T-cells to pass the 'E' to 'L' transition correctly during ßselection (Schmidt, 1998a).

Gfi-1 is a cellular proto-oncogene that has been identified as a target of provirus integration in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced lymphomas. Gfi-1 encodes a zinc finger protein that functions as a transcriptional repressor. Gfi-1B, a Gfi-1 related gene expressed in bone marrow and spleen, also encodes a transcriptional repressor. Both IL-6-induced G1 arrest and differentiation of the myelomonocytic cell line M1 are linked to the downregulation of Gfi-1B and the parallel induction of the cyclin-dependent kinase inhibitor p21WAF1. Experiments addressing the potential mechanism of the apparent coordinate regulation of these genes reveal that Gfi-1B represses p21WAF1 directly by binding to a high-affinity site at -1518 to -1530 in the p21WAF1 promoter. Forced expression of Gfi-1B, but not of Gfi-1B deletion mutants lacking the repressor domain, blocks the IL-6-mediated induction of p21WAF1 and inhibits G1 arrest and differentiation. It is concluded that Gfi-1B is a direct repressor of the p21WAF1 promoter, the first such repressor identified to date, and that sustained expression of Gfi-1B blocks IL-6-induced G1 arrest and differentiation of M1 cells perhaps because it prevents p21WAF1 induction by IL-6 (Tong, 1998).

STAT factors act as signal transducers of cytokine receptors and transcriptionally activate specific target genes. The recently discovered protein PIAS3 binds directly to STAT3 and blocks transcriptional activation. Experimental evidence is presented implementing the zinc finger protein Gfi-1 as a new regulatory factor in STAT3-mediated signal transduction. The interaction between the two proteins first became evident in a yeast two-hybrid screen but is also seen in coprecipitation experiments from eukaryotic cells. Moreover, both Gfi-1 and PIAS3 colocalize in a characteristic nuclear dot structure. While PIAS3 exerts a profound inhibitory effect on STAT3-mediated transcription of target promoters, Gfi-1 can overcome the PIAS3 block and significantly enhances STAT3-mediated transcriptional activation. In primary T cells, Gfi-1 is able to amplify IL-6-dependent T-cell activation. Since Gfi-1 is a known, dominant proto-oncogene, these findings bear particular importance for the recently described ability of STAT3 to transform cells malignantly and offers an explanation of the oncogenic potential of Gfi-1 in T lymphocytes (Rodel, 2000).

Gfi-1 is a nuclear zinc finger protein with the activity of a transcriptional repressor and the ability to predispose for the development of T-cell lymphoma when expressed constitutively at high levels. Whereas thymic T-cell precursors express endogenous Gfi-1, mature peripheral T-cells lack Gfi-1 but upregulate its expression transiently after antigenic stimulation and activation of Erk1/2 demonstrating a role of Gfi-1 in T-cell activation. Constitutive expression of Gfi-1 accelerates S phase entry of primary, resting T-cells upon antigenic stimulation. In addition, high level Gfi-1 expression inhibits phorbol ester induced G1 arrest and activation induced cell death in Jurkat T-cells. These effects of Gfi-1 concur with lower absolute levels and hyperphosphorylation of the pocket protein pRb. Moreover, phorbol ester induced expression of the negative cell cycle regulator p21(WAF1) is blocked in the presence of Gfi-1. These findings suggest that Gfi-1 contributes to T-cell lymphomagenesis by overriding a late G1 cell cycle checkpoint that controls activation induced death and S phase entry of T-cells (Karsunky, 2002).

SOCS proteins take part in a classical negative feedback loop to attenuate cytokine signaling. Although STAT family members positively modulate Socs gene expression, little else is known about Socs gene regulation. This study identifies functional binding sites for GFI-1B, a proto-oncogenic transcriptional repressor, in the promoters of murine Socs1 and Socs3. Thus, mutating these sites relieved transcriptional repression, as determined by luciferase reporter assays of transiently transfected erythropoietin-responsive 32D-EpoR and HCD57 cells. Furthermore, cotransfection of Gfi-1B expression plasmid repressed reporter activity of wild-type (but not mutagenized) Socs1 and Socs3 promoters, strongly suggestive of direct GFI-1B binding to these promoters. In addition, overexpression of Gfi-1B resulted in reduced transcript levels of Socs1 and Socs3, but not Socs2 or Cis. Upon stimulation with erythropoietin, Socs transcripts are rapidly induced, whereas Gfi-1B mRNA is down-regulated. Interestingly, the latter effect appears to rely on STAT5 activity, but not on phosphoinositide 3-kinase or MAPK pathways. Thus, cytokine-mediated STAT5 activation allows relief of direct repression by GFI-1B of the Socs1 and Socs3 promoters, but apparently not of the Socs2 and Cis promoters. This constitutes a previously undescribed mode of controlling cytokine responsiveness, through the direct repression of a tumor suppressor (SOCS1) by a proto-oncoprotein (GFI-1B) (Jegalian, 2002).

Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. Human beta1 sGC transcriptional regulation was investigated in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, this study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression (Sharina, 2003).

Granulocyte-colony-stimulating factor (G-CSF) stimulates the activation of multiple signaling pathways, leading to alterations in the activities of transcription factors. Gfi-1 is a zinc finger transcriptional repressor that is required for granulopoiesis. How Gfi-1 acts in myeloid cells is poorly understood. The expression of Gfi-1 is up-regulated during G-CSF-induced granulocytic differentiation in myeloid 32D cells. Truncation of the carboxyl terminus of the G-CSF receptor, as seen in patients with acute myeloid leukemia evolving from severe congenital neutropenia, disrupts Gfi-1 up-regulation by G-CSF. Ectopic expression of a dominant negative Gfi-1 mutant, N382S, which is associated with severe congenital neutropenia, results in premature apoptosis and reduces proliferation of cells induced to differentiate with G-CSF. The expression of neutrophil elastase (NE) and CCAAT enhancer-binding protein epsilon (C/EBPepsilon) is significantly increased in 32D cells expressing N382S. In contrast, overexpression of wild type Gfi-1 abolishes G-CSF-induced up-regulation of C/EBPepsilon but has no apparent effect on NE up-regulation by G-CSF. Notably, G-CSF-dependent proliferation and survival are inhibited upon overexpression of C/EBPepsilon but not NE. These data indicate that Gfi-1 down-regulates C/EBPepsilon expression and suggest that increased expression of C/EBPepsilon as a consequence of loss of Gfi-1 function may be deleterious to the proliferation and survival of early myeloid cells (Zhuang, 2006).

Gfi-1 mutation

Gfi-1 and Gfi-1b, homologs of Drosophila senseless, are novel proto-oncogenes identified by retroviral insertional mutagenesis. By gene targeting, it has been established that Gfi-1b is required for the development of two related blood lineages, erythroid and megakaryocytic, in mice. Gfi-1b^-/- embryonic stem cells fail to contribute to red cells of adult chimeras. Gfi-1b^-/- embryos exhibit delayed maturation of primitive erythrocytes and subsequently die with failure to produce definitive enucleated erythrocytes. The fetal liver of mutant mice contains erythroid and megakaryocytic precursors arrested in their development. Myelopoiesis is normal. Therefore, Gfi-1b is an essential transcriptional regulator of erythroid and megakaryocyte development (Saleque, 2002).

The mammalian protein Gfi-1b, like its orthologs in Drosophila and C. elegans, Senseless and PAG-3 respectively, regulates the development of specific cellular lineages. The three proteins also show similar DNA-binding specificities consistent with >80% sequence identity between their DNA-binding zinc fingers, and presumably have similar target sites in vivo. However, whether they regulate analogous target genes and pathways in vivo remains to be elucidated. Notably, both PAG-3 and Senseless lack the SNAG repression domain, and this structural variation could lead to mechanistic differences between them in regulating their targets. The control of sensory organ development in Drosophila by an autoregulatory loop comprised of senseless and the basic-helix-loop-helix (bHLH) proneural genes daughterless, achaete-scute, and atonal raises the possibility that Gfi-1b may also interact in a transcriptional network with bHLH factors, within or outside the hematopoietic system. A likely candidate within the hematopoietic system is the bHLH factor SCL/tal-1, a gene required for development of all hematopoietic lineages. Because loss of Gfi-1b does not affect SCL/tal-1 expression in fetal liver colonies, it is concluded that Gfi-1b is not required for SCL expression. Whether SCL regulates Gfi-1b expression is unknown (Saleque, 2002).

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosoophila and pag-3 in C. elegans are expressed and required in the nervous system. This study shows that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies: they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death (Wallis, 2003).

Gfi1 is a transcriptional repressor implicated in lymphomagenesis, neutropenia, and hematopoietic development, as well as ear and lung development. This study demonstrates that Gfi1 functions downstream of Math1 in intestinal secretory lineage differentiation. Gfi1^-/- mice lack Paneth cells, have fewer goblet cells, and supernumerary enteroendocrine cells. Gfi1^-/- mice show gene expression changes consistent with this altered cell allocation. These data suggest that Gfi1 functions to select goblet/Paneth versus enteroendocrine progenitors. A model of intestinal cell fate choice is proposed in which beta-catenin and Cdx function upstream of Math1, and lineage-specific genes such as Ngn3 act downstream of Gfi1 (Shroyer, 2005).

Gfi-1 is a zinc finger transcriptional repressor originally recognized for its role in T cell differentiation and lymphomas. Recent experiments reveal that gene-targeted Gfi-1-deficient mice are neutropenic and that Gfi-1 mutations cause human neutropenia. In both cases, myeloid progenitor cells lose the ability to distinctly differentiate granulocytes from monocytes. The molecular mechanism of the hematopoietic abnormalities caused by Gfi-1 deficiency remains undetermined because of a lack of known Gfi-1 target genes. To identify Gfi-1 targets in vivo, large-scale chromatin immunoprecipitation analysis was performed on a set of 34 candidate genes in myeloblast (KG-1 and HL-60), monoblast (U937), and T lymphocyte cell lines (Jurkat), in concert with RT-PCR-based expression profiling. Thirty-two Gfi-1 binding sites were identified in a functionally variable set of 16 genes, including complements of cell-cycle regulators, transcription factors, and granulocyte-specific markers. Cluster analysis of expression patterns and chromatin immunoprecipitation data reveals that Gfi-1 targets a subset of genes differentiating hematopoietic lineages and therefore plays a relatively superior role in the hierarchy of factors governing stem cell differentiation (Duan, 2003).

Transcriptional regulation of Gfi-1

Expression of Gfi (growth factor-independence)-1B, a Gfi-1-related transcriptional repressor, is restricted to erythroid lineage cells and is essential for erythropoiesis. The transcription start site of the human Gfi-1B gene has been determined and its first non-coding exon has been located approximately 7.82 kb upstream of the first coding exon. The genomic sequence preceding this first non-coding exon has been identified to be its erythroid-specific promoter region in K562 cells. Using gel-shift and chromatin immunoprecipitation (ChIP) assays, it has been demonstrated that NF-Y and GATA-1 directly participate in transcriptional activation of the Gfi-1B gene in K562 cells. Ectopic expression of GATA-1 markedly stimulates the activity of the Gfi-1B promoter in a non-erythroid cell line U937. Interestingly, these results have indicated that this GATA-1-mediated trans-activation is dependent on NF-Y binding to the CCAAT site. It is concluded that functional cooperation between GATA-1 and NF-Y contributes to erythroid-specific transcriptional activation of Gfi-1B promoter (Huang, 2004)

Gfi1b is a 37 kDa transcriptional repressor with six zinc-finger domains that is differentially expressed during hemato- and lymphopoiesis. Transcription from the Gfi1b gene locus is silenced in the spleen but not in the bone marrow of transgenic mice that constitutively express Gfi1b under the control of the pan-hematopoietic vav promoter. Sequence analysis of the Gfi1b promoter showed the presence of potential Gfi1/Gfi1b-binding sites close to the mRNA start site. The expression of reporter gene constructs containing the Gfi1b core promoter appended to the luciferase gene were strongly repressed in the presence of exogenous Gfi1b. Moreover, analysis of combinatorial mutant mice that carry the vav-Gfi1b transgene and a green fluorescent protein-tagged Gfi1 gene locus demonstrated that the Gfi1 gene can be repressed by Gfi1b. Direct binding of Gfi1b and Gfi1 to the potential binding sites in the Gfi1b promoter could be demonstrated by gel-shift analyses in vitro. Chromatin-immunoprecipitation experiments showed that both the Gfi1b and the Gfi1 promoter are indeed occupied by Gfi1b in vivo. Hence, it is conclude that Gfi1b can auto-repress its own expression, but, in addition, is also able to cross-repress expression of the Gfi1 gene most likely in a cell type specific manner (Vassen, 2005).

Gfi-1 repression of transcription

Gfi-1 and Gfi-1B can repress transcription and play important roles in hematopoietic cell survival and differentiation. Although these proteins are known to bind DNA through a C-terminal zinc-finger domain and may require an N-terminal SNAG domain (SNAIL/Gfi-1) to repress transcription, the mechanism by which Gfi-1 and Gfi-1B act is unknown. A first step towards understanding the mechanism by which these proteins repress transcription is to identify interacting proteins that could contribute to transcriptional repression. ETO (also termed MTG8), was first identified through its involvement in the (8;21) translocation associated with acute myelogenous leukemia. It attaches to the nuclear matrix and associates with histone deacetylases and the co-repressors N-CoR, SMRT, and mSin3A, and may act as a co-repressor for site-specific transcriptions factors. Gfi-1 interacts with ETO and related proteins both in vitro and in vivo and with histone deacetylase proteins in vivo. A portion of Gfi-1 and Gfi-1B associates with the nuclear matrix, as is the case with ETO. Moreover, Gfi-1 and ETO co-localize to punctate subnuclear structures. When co-expressed in mammalian cells, Gfi-1 associates with histone deacetylse-1 (HDAC-1), HDAC-2, and HDAC-3. These data identify ETO as a partner for Gfi-1 and Gfi-1B, and suggest that Gfi-1 proteins repress transcription through recruitment of histone deacetylase-containing complexes (McGhee, 2003).

Growth factor independence-1 (GFI1) and GFI1B are closely related, yet differentially expressed transcriptional repressors with nearly identical DNA binding domains. GFI1 is upregulated in the earliest thymocyte precursors, while GFI1B expression is restricted to T lymphopoiesis stages coincident with activation. Transgenic expression of GFI1 potentiates T-cell activation, while forced GFI1B expression decreases activation. Both mice and humans with mutant Gfi1 display lymphoid abnormalities. This study describes autoregulation of Gfi1 in primary mouse thymocytes and a human T-cell line. GFI1 binding to cis-element sequences conserved between rat, mouse and human Gfi1 mediates direct and potent transcriptional repression. In addition, dramatic regulation of Gfi1 can also be mediated by GFI1B. These data provide the first example of a gene directly targeted by GFI1 and GFI1B. Moreover, they support a role for auto- and trans-regulation of Gfi1 by GFI1 and GFI1B in maintaining the normal expression patterns of Gfi1, and suggest that GFI1B may indirectly affect T-cell activation through repression of Gfi1 (Doan, 2004).

Gfi-1 and hematopoesis

In the search for genes expressed in hematopoietic stem cells, the expression of Gfi-1B (growth factor independence-1B) was found to be highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and 6 zinc finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, its function was analyzed in human hematopoiesis. Enforced expression of Gfi-1B in human CD34(+) hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression of Gfi-1B does not promote erythroid commitment, but enhances proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biologic effects of Gfi-1B are mediated through its zinc finger domain, but not by the SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription is dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression (Osawa, 2002).

Mammalian Gfi-1 is a proto-oncogene

During progression of Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphomas, growth selection results in the expansion of cell clones carrying increasing numbers of integrated proviruses. These new provirus insertions reproducibly contribute to enhanced growth, allowing the emergence of cell clones from the initially heterogeneous population of tumor cells. The Mo-MuLV-induced rat T cell lymphoma lines 2780d and 5675d, which are dependent on interleukin-2 (IL-2) for growth in culture (IL-2d), were placed in IL-2-free medium to select for IL-2-independent (IL-2i) mutants. Southern blot analysis of genomic DNA from these mutants, which was hybridized to a Mo-MuLV long terminal repeat probe, reveals that all mutants carry new provirus insertions (from one to four new proviruses per cell line). A locus of integration identified through cloning of the single new provirus detected in one of the IL-2i mutants, 2780i.5, has been found to be the target of provirus insertion in 1 additional IL-2i cell line, of the 24 tested. A full-length cDNA of a gene (growth factor independence-1 [Gfi-1]) activated by promoter insertion in the 2780i.5 cells was cloned and shown to encode a novel zinc finger protein. Gfi-1 is expressed at low levels in IL-2d cell lines cultured in IL-2-containing medium and at high levels in most IL-2i cell lines, including the two harboring a provirus at this locus. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated splenocytes, Gfi-1 expression begins to rise at 12 h after stimulation and reaches very high levels after 50 h, suggesting that it may be functionally involved in events occurring after the interaction of IL-2 with its receptor, perhaps during the transition from the G1 to the S phase of the cell cycle. In agreement with this, Gfi-1 does not induce the expression of IL-2. Expression of Gfi-1 in 2780d cells following transfer of a Gfi-1/LXSN retrovirus construct contributes to the emergence of the IL-2i phenotype (Gilks, 1993).

The clonality of lymphomas that originate in myc/pim-1 bitransgenic mice due to synergistic action of both oncogenes indicates the requirement of additional events for progression to full malignancy. To isolate genes that cooperate with both myc and pim-1, provirus tagging with E mu L-myc/pim-1 double transgenic mice were used. Accelerated tumor formation is found in infected animals and the gfi-1 gene and neighboring loci on mouse chromosome 5 are occupied by proviruses in about 53% of the tumors leading in all cases to high level gfi-1 expression. Forced expression of the gfi-1 encoded zinc finger protein in IL-2 dependent T-cells provokes increased survival upon IL-2 depletion and evidence is presented that this occurs at least in part through stimulation of proliferation. The data suggest that gfi-1 is a proto-oncogene cooperation with both myc and pim-1 genes in T-cell lymphomagenesis (Zornig, 1996).

The Gfi-1 protooncogene encodes a nuclear zinc-finger protein that carries a novel repressor domain, SNAG, and functions as a position- and orientation-independent active transcriptional repressor. The Gfi-1 repressor allows interleukin 2 (IL-2)-dependent T cells to escape G1 arrest induced by IL-2 withdrawal in culture and collaborates with c-myc and pim-1 for the induction of retrovirus-induced lymphomas in animals. Overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL-2-deficient media. Similarly, induction of Gfi-1 in primary thymocytes from mice carrying a metal-inducible Gfi-1 transgene inhibits cell death induced by cultivation in vitro. The protein and mRNA levels of the proapoptotic regulator Bax are down-regulated by Gfi-1 in both immortalized T-cell lines and primary transgenic thymocytes. The repression is direct and depends on several Gfi-1-binding sites in the p53-inducible Bax promoter. In addition to Bax, Gfi-1 also represses Bak, another apoptosis-promoting member of the Bcl-2 gene family. Therefore, Gfi-1 may inhibit apoptosis by means of its repression of multiple proapoptotic regulators. The antiapoptotic properties of Gfi-1 provide a potential explanation for its strong collaboration with c-myc during oncogenesis (Grimes, 1996b).

A prominent feature of retrovirus-induced immunodeficiency in mice (MAIDS) is early polyclonal activation of CD4+ T cells followed by the appearance of monoclonal lymphomas marked by clonal proviral integrations. These events appear to occur independent of interleukin-2 (IL-2), suggesting the activity of an alternative growth-promoting pathway. The possible contributions to T cell expansion of a gene, Gfi-1, previously shown to confer IL-2 independence to rat T cell lymphomas, was investigated. Seventeen mice with MAIDS that had clonal populations of T cells were studied. Proviral integrations at Gfi-1 were detected in two animals. These integrations were associated with enhanced transcription of Gfi-1. Unexpectedly, elevated levels of Gfi-1 transcripts were also observed in four T cell lymphomas without detectable integrations at this locus. This suggests that IL-2-independent T cell growth in MAIDS may be driven by transcriptional activation of Gfi-1 by proviral insertion or transactivation (Liao, 1997).

The gfi-1 gene encodes a zinc finger containing protein that is specifically expressed in T-lymphocytes and is a frequent target of proviral insertion in T-cell lymphoma provoked by infection with MoMuLV -- a non acute transforming retrovirus. Expression of a gfi-1 transgene targeted to T-cells by the lck proximal promoter provokes a reduction of peripheral CD4 and CD8 positive T-cells but nevertheless weakly predisposes transgenic animals for the development of T-cell lymphoma. Forced coexpression of the serine/threonine kinase Pim-1 can partially restore normal T-cell numbers in double pim-1/gfi-1 transgenic mice. Moreover, the combinatorial expression of Pim-1 and Gfi-1 leads to accelerated development of T-cell lymphoma with a mean latency period of 114 days. A similar accelerated rate of lymphoma development is observed when lck-gfi-1 mice were crossed with mice that carry a L-myc gene targeted to be expressed at high levels in T-cells. The results show that gfi-1 can act with low activity as a dominant oncogene when overexpressed but also demonstrate that it is most efficient only in the presence of a cooperative partner protein, as for example Pim-1 or L-Myc. In addition, the results suggest that Pim-1 and Gfi-1 are acting synergistically in both T-cell lymphomagenesis and T-cell development (Schmidt, 1998b).

This study reports essential roles of zinc finger transcription factor Gfi-1 in myeloid development. Gene-targeted Gfi-1^-/- mice lack normal neutrophils and are highly susceptible to abscess formation by gram-positive bacteria. Arrested, morphologically atypical, Gr1⁺Mac1⁺ myeloid cells expand with age in the bone marrow. RNAs encoding primary but not secondary or tertiary neutrophil (granulocyte) granule proteins are expressed. The atypical Gr1⁺Mac1⁺ cell population shares characteristics of both the neutrophil and macrophage lineages and exhibits phagocytosis and respiratory burst activity. Reexpression of Gfi-1 in sorted Gfi-1^-/- progenitors ex vivo rescues neutrophil differentiation in response to G-CSF. Thus, Gfi-1 not only promotes differentiation of neutrophils but also antagonizes traits of the alternate monocyte/macrophage program (Hock, 2003).

Haematopoietic stem cells (HSCs) sustain blood production throughout life. HSCs are capable of extensive proliferative expansion; a single HSC may reconstitute lethally irradiated hosts. In steady-state, HSCs remain largely quiescent and self-renew at a constant low rate, forestalling their exhaustion during adult life. Whereas nuclear regulatory factors promoting proliferative programmes of HSCs in vivo and ex vivo have been identified, transcription factors restricting their cycling have remained elusive. This study reports that the zinc-finger repressor Gfi-1 (growth factor independent 1), a cooperating oncogene in lymphoid cells, unexpectedly restricts proliferation of HSCs. After loss of Gfi-1, HSCs display elevated proliferation rates as assessed by 5-bromodeoxyuridine incorporation and cell-cycle analysis. Gfi-1^-/- HSCs are functionally compromised in competitive repopulation and serial transplantation assays, and are rapidly out-competed in the bone marrow of mouse chimaeras generated with Gfi-1^-/- embryonic stem cells. Thus, Gfi-1 is essential to restrict HSC proliferation and to preserve HSC functional integrity (Hock, 2004).

Human small cell lung cancers might be derived from pulmonary cells with a neuroendocrine phenotype. They are driven to proliferate by autocrine and paracrine neuropeptide growth factor stimulation. The molecular basis of the neuroendocrine phenotype of lung carcinomas is relatively unknown. The Achaete-Scute Homologue-1 (ASH1) transcription factor is critically required for the formation of pulmonary neuroendocrine cells and is a marker for human small cell lung cancers. The Drosophila orthologues of ASH1 (Achaete and Scute) and the growth factor independence-1 (GFI1) oncoprotein (Senseless) genetically interact to inhibit Notch signaling and specify fly sensory organ development. This study shows that GFI1, as with ASH1, is expressed in neuroendocrine lung cancer cell lines and that GFI1 in lung cancer cell lines functions as a DNA-binding transcriptional repressor protein. Forced expression of GFI1 potentiates tumor formation of small-cell lung carcinoma cells. In primary human lung cancer specimens, GFI1 expression strongly correlates with expression of ASH1, the neuroendocrine growth factor gastrin-releasing peptide, and neuroendocrine markers synaptophysin and chromogranin A. GFI1 colocalizes with chromogranin A and calcitonin-gene-related peptide in embryonic and adult murine pulmonary neuroendocrine cells. In addition, mice with a mutation in GFI1 display abnormal development of pulmonary neuroendocrine cells, indicating that GFI1 is important for neuroendocrine differentiation (Kazanjian, 2004).

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date revised: 22 June 2023

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