daughterless

Transcriptional Regulation

The cell surface receptor Notch is required during development of Drosophila melanogaster for differentiation of numerous tissues. Notch is often required for specification of precursor cells by lateral inhibition and subsequently for differentiation of tissues from these precursor cells. Certain embryonic cells and tissues that develop after lateral inhibition, like the connectives and commissures of the central nervous system, are enriched for a form of Notch not recognized by antibodies made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats. Western blotting and immunoprecipitation analyses show that Notch molecules lacking this region are produced during embryogenesis and form protein complexes with the ligand Delta. Experiments with cultured cells indicate that Delta promotes accumulation of a Notch intracellular fragment lacking the carboxyl terminus. Furthermore, Notch that lacks the carboxyl terminus functions as a receptor for Delta. These results suggest that Notch activities during development include generation and activity of a truncated receptor designated NdeltaCterm (Wesley, 2000).

The regulation of da expression by NdeltaCterm may be significant for embryogenesis. da genetically interacts with Notch; it is required for development of the nervous system from neuroblasts but not for lateral inhibition, and the Daughterless protein promotes DNA-binding activities of the proneural Achaete-Scute Complex proteins. Both NdeltaCterm and Daughterless protein accumulate in segregating neuroblasts raising the possibility that NdeltaCterm is involved in this upregulation of da expression. Accordingly, nd³ embryos that overproduce NdeltaCterm also overproduce da RNA in the neuroblasts (Wesley, 2000).

In the embryo, da is expressed at low levels in almost all cells but is upregulated in certain cells including the segregating neuroblasts. S2 cells expressing NFull and NdeltaCterm receptors have lower levels of DA RNA than S2 cells without N. In response to Dl, only S2-NdeltaCterm cells increase expression of DA RNA, but only to the level observed in cells without N. Therefore, it appears possible that with the expression of different forms of N, developing cells acquire an ability to differentially regulate the otherwise constitutive da expression. Such differential regulation might be important for suppressing the activities of Achaete-Scute Complex proteins in the developing epidermis where NFull is expected to function, but not in the developing nervous system where NdeltaCterm is expected to function. Since both N receptors have the ability to activate E(spl)C, the timing and sequence of expression of NFull and NdeltaCterm may also be important for development (Wesley, 2000).

As the only class I helix-loop-helix transcription factor in Drosophila, Daughterless (Da) has generally been regarded as a ubiquitously expressed binding partner for other developmentally regulated bHLH transcription factors. From analysis of a novel tissue-specific allele, da^lyh, da expression has been shown to not be constitutive, but rather, is dynamically regulated. This transcriptional regulation includes somatic ovary-specific activation, autoregulation and negative regulation. Unexpectedly, the diverse functions of da may require that expression levels be tightly controlled in a cell and/or tissue-specific manner. This analysis of da^lyh identifies it as the first springer insertion (the springer element is an 8.8kD retrotransposon) that functions as an insulating element, with its disruptive activity mediated by the product of a fourth chromosome gene, Suppressor of lyh [Su(lyh)] (Smith, 2001).

The helix-loop-helix (HLH) family of transcription factors includes over 240 different proteins that are present throughout eukaryotes, including both ubiquitous and temporally/spatially restricted transcription factors. HLH proteins dimerize via amphipathic helices and interact directly with the major groove of DNA via a basic domain. These proteins fall into seven specific classes based on dimerization capabilities, tissue distribution and DNA target specificity. Class I HLHs, also known as E proteins, can form either heterodimers or homodimers, are widely expressed, and have DNA binding specificity for the E box. The more numerous class II HLHs heterodimerize with class I HLHs and show tissue-restricted expression patterns and target sequence specificity that varies with different heterodimer partners and their conformation. In vertebrates, class I HLHs are essential for commitment to the B lymphoid lineage, T cell development, regulation of V(D)J recombination, muscle differentiation and expression of differentiated cell products such as insulin. This partial list of vertebrate class I HLH functions does not include cases of transcriptional regulation for which a class II HLH is known (Smith, 2001 and references therein).

There are at least two well-described requirements for da function in the ovary: in the germline for progeny sex determination (Cronmiller, 1987) and in the somatic ovary for follicle formation (Cummings, 1994). The functional unit of the ovary is the ovariole, and within the germarium of each ovariole, two distinct stem cell populations give rise to either germline or soma. A germline stem cell divides asymmetrically to produce a cystoblast, which undergoes four rounds of mitotic divisions with incomplete cytokinesis to produce an oocyte with its 15 interconnected nurse cells. It is in these germline cells that da mRNA is produced and eventually concentrated into the oocyte for the maternal sex determination function; da germline mRNA does not appear to be translated during oogenesis. As the cyst moves posteriorly through the germarium, the somatic stem cells give rise to somatic cells that (1) envelop the cyst to form a follicle and (2) form stalks that separate adjacent follicles. Da protein is found within these somatic cells, where it is required for proper encapsulation and separation of follicles (Smith, 2001 and references therein).

The female sterile mutant, fs(2)lyh, which arose spontaneously in an enhancer trap screen for genes expressed during oogenesis, is an allele of da that specifically disrupts function of the gene within the somatic ovary. In complementation tests with da alleles and genetic interaction tests with Sxl, sis-a and sis-b, fs(2)lyh shows no maternal effects on sex determination. Similarly, fs(2)lyh is fully viable and shows no visible phenotypes in combination with null da alleles and deletions, demonstrating that no other da functions are disrupted. However, fs(2)lyh fails to complement da alleles for follicle formation: fs(2)lyh/fs(2)lyh and fs(2)lyh/da^- ovaries have multiple indistinguishable follicular defects, such as missing stalks, multicyst follicles and late stage necrosis. In both cases, the phenotype worsens with age. Based on the mutant ovary morphology, fs(2)lyh behaves like a null allele: there is no discernible difference in mutant phenotypes from flies of equal ages homozygous for fs(2)lyh or transheterozygous for either a da null allele or a deletion, even in the youngest flies that exhibit the least extreme phenotype. Henceforth, fs(2)lyh will be referred to as da^lyh (Smith, 2001).

Da protein expression in da^lyh mutants is also disrupted specifically in the ovary. Staining of wild-type ovaries shows clear nuclear Da protein, while da^lyh mutant ovaries show no nuclear localized protein within the ovariole. Western blots of ovary extracts detect Da protein but at reduced levels. The protein seen in ovary extract probably corresponds to other Da-containing tissues included in the extract: the epithelial sheath that surrounds each ovariole and its associated muscles, the tracheae that infiltrate the ovary and the oviduct (Smith, 2001).

The molecular lesion in da^lyh is caused by the insertion of a springer element. Southern blot analysis of the da region indicates the insertion of approximately 8 kb of DNA. PCR amplification, cloning and sequencing of the inserted DNA identified the insertion as a gypsy-like springer retrotransposon. The partial sequence data together with that of the Berkeley Drosophila Genome Project, which includes several full-length copies of the springer element, has confirmed the strong similarity between springer and gypsy: homologous ORFs are 36% to 65% identical. The da^lyh springer inserted 113 bp into the da intron, produces a characteristic TATA target site duplication (Smith, 2001).

The mechanism of da gene disruption by the springer element is different from other analyzed springer alleles. Molecularly characterized springer-induced mutations (Tm2³, N^fa3, f^36a, Mhc², Mhc³) each result from insertion within exons or near alternatively used exons to produce aberrant transcripts usually with premature transcriptional termination within the springer LTR. The da^lyh mutation increases the da transcript levels. da transcript levels are 1.5- and 1.9-fold higher (males and females, respectively) in da^lyh than in wild-type flies. Taken together, the RNA and protein analyses suggest that the da^lyh springer insertion acts as a transcriptional insulator. Additionally, the springer element appears to insulate a negative regulatory element that results in an overall increase in da transcription, hence the elevated mRNA levels evident on the Northern blots (Smith, 2001).

Surprisingly, in genetic interaction tests da^lyh does not behave like a genetic null: ordinarily, the da somatic ovary function is particularly sensitive to gene dose, such that a da loss-of-function allele exhibits second site non-complementation with mutations in other genes involved in follicle morphogenesis. By contrast, da^lyh does not. A specific example of this paradoxical genetic behavior is the interaction observed between da and stall (stl), another gene required for follicle formation. A null allele of da completely fails to complement the null allele, stl^a16; ovaries of doubly heterozygous females have no normal ovarioles. Even the hypomorphic da^s22 allele fails to complement stl^a16, with 65% of the ovarioles having defects. However, da^lyh fully complements stl^a16; no defects are seen. Thus, the da⁺ chromosome of the heterozygous da^lyh genotype appears to induce wild-type function from its mutant da^lyh homolog, and does not produce Da protein in the somatic ovary (Smith, 2001).

The genetic evidence for da autoregulation was validated by demonstrating molecularly that Da protein could transactivate the da promoter in vivo. The genetic transactivation of da^lyh that is observed reflects a normal activity of Da protein on the wild-type da promoter, and in da^lyh this autoregulation is not insulated by the springer element of this mutant allele (Smith, 2001).

The fact that the da^lyh phenotypes result from insulating properties of the springer insertion of this mutant is strengthened by the identification of dominant suppressor mutations in another gene. Four alleles of Suppressor of lyh (Su(lyh)) were isolated in a screen for rescue of da^lyh sterility. The strongest allele rescues da^lyh female sterility completely, with only 15% of the ovarioles still showing mild defects. In a homozygous Su(lyh) background, da^lyh ovaries are completely normal. By contrast, Su(lyh) has no effect on the mutant ovary phenotype associated with other da alleles: it does not suppress da7/das22 ovary defects. The specificity of Su(lyh) for da^lyh suggests that the Su(lyh) gene product is required for the insulating properties of the springer insertion that results in the da^lyh loss-of-function phenotype. The same Su(lyh) gene activity could account for the da^lyh-associated overexpression phenotype, since the Su(lyh) mutant also acts as a dominant suppressor of that phenotype. No phenotype associated with the Su(lyh) mutations could be detected in an otherwise wild-type background; thus, the wild-type function of this gene is unclear (Smith, 2001).

Thus, despite the expectation that a ubiquitously expressed binding partner for other developmentally regulated proteins would have a simple constitutive promoter, da is under precise transcriptional control. da is transcriptionally autoregulated, and both positive and negative cis-acting regulatory sites are involved. A model is suggested to describe the control of wild-type da expression in the somatic ovary. This model also accounts for all aspects of the da^lyh misregulation. In wild-type flies, several distinct transcriptional controls provide for a tightly regulated level of da expression within the somatic ovary. Initiation of da transcription requires an enhancer within the single intron of the gene. After activation of da transcription, the Da protein itself functions to maintain da expression. Indeed, since multiple canonical E-boxes are present in the da promoter region, this autoregulatory function may result from a direct interaction of Da protein with its own regulatory sequences. Finally, negative cis-acting sequences downregulate da transcription, thus preventing autoregulatory da expression from escalating to produce deleteriously high levels of Da protein. Such deleterious levels are achieved by overexpression of heat shock inducible da⁺ transgenes (Smith, 2001).

The insertion of the springer retrotransposon in the da^lyh mutant impacts the cis-acting transcriptional regulation of da, both positively and negatively, without disrupting autoregulation. By insulating the da promoter from the intronic enhancer sequences, the springer insertion effectively blocks activation of da transcription in the somatic ovary. Thus, the da^lyh homozygote exhibits a da null phenotype in the ovary. By contrast, the mutant allele of a da^lyh heterozygote is functional: Da protein derived from expression of the wild-type allele transactivates the mutant allele, thus bypassing the need for a functional enhancer. In this way, transactivation of da^lyh by da⁺ accounts for the wild-type behavior of da^lyh heterozygotes in genetic interaction tests. The springer retrotransposon also insulates the da promoter from the negative cis-acting sequences that must lie downstream of the insertion site. In da^lyh homozygotes, in addition to the loss of da transcription in the somatic ovary, there is an overall increase in da transcript levels. Furthermore, in da^lyh homozygotes carrying an extra copy of da⁺, the wild-type allele transactivates the mutant alleles, resulting in excessive da transcription and an associated gain-of-function phenotype in the ovary (Smith, 2001).

Based on the failure of transcription of da^lyh in the somatic ovary and from an analysis of a da promoter fusion transgene, a STAT (Drosophila Stat92E)-binding site has been identified as a candidate for the cis-acting enhancer. STAT (signal transducers and activators of transcription) proteins are activated by tyrosine kinases in response to cytokine or growth factor signals and play essential developmental roles in growth and differentiation, and they are constitutively activated in many cancers. The Stat92E binding site in da (TTCATGGAA) is the only predicted transcription factor-binding site found exclusively downstream of the springer insertion and within the extents of the da.G32 reporter. The somatic ovary enhancer must be included in the da.G32 reporter, since this transgene is expressed in the ovary, even in da^lyh mutants. Moreover, a temperature sensitive loss-of-function allele of Stat92E shows a da-like mutant ovary phenotype. It is proposed that Stat92E is essential for the initiation of da transcription within the somatic ovary (Smith, 2001).

Da protein appears to be necessary for maintenance of its own transcription, and the simplest molecular model for da autoregulation is direct transcriptional activation. Although Da homodimers can bind DNA in vitro and the mammalian homolog, E47, does function as a homodimer in B cell development, there are no examples of Da protein homodimerizing to activate transcription in vivo. More likely, Da acts on its own promoter by collaboration with another bHLH-binding partner. Several possible candidate binding partners, based on ovary phenotypes in genetic interaction tests, have been identified. For example, one candidate is achaete (ac), which transcriptionally autoregulates during the development of sensory bristles in wing imaginal discs. For this process, Ac protein heterodimerizes with Da; perhaps they collaborate again in the somatic ovary with the da gene as their target (Smith, 2001).

Two cis-acting elements involved in da transcriptional regulation in the ovary have been identified, but there are likely to be more cis-regulatory elements whose use may differ between or within other tissues. Although nearly ubiquitous throughout development, Da protein is present at significantly different levels in various tissues, or even within individual tissues. For example, the CNS includes cells with levels of Da that range from very low to very high, and in eye discs, dynamic changes in Da protein levels correlate with the progression of the morphogenetic furrow. If Da protein levels directly reflect da transcript levels, these observations suggest that precise regulation of da is crucial for developmental processes, and the regulatory sites identified are probably not sufficient to account for the scope of regulation necessary. The da.G32 reporter, which shows a mottled expression pattern that is not attributable to position effect variegation, indicates that this transgene is missing crucial binding sites for regulatory factors. Additionally, this construct is unable to rescue embryonic lethality when driving a Gal4-dependent da⁺ transgene. However, a 15 kb genomic da⁺ transgene that includes the da.G32 regulatory region and an additional 12 kb downstream rescues da mutant flies to adulthood (H. Vaessin, personal communication to Smith, 2001); this construct may contain all of the necessary da regulatory sequences. Thus, da expression, like that of many other protein-coding genes, is dependent upon a balance of multiple positive and negative regulators (Smith, 2001).

The da^lyh allele is the first springer-induced mutation in which this retrotransposon is documented to disrupt gene function by acting as an insulator; this discovery emphasizes the similarity between springer and the extensively-characterized gypsy retrotransposon. Like gypsy, springer can disrupt gene function in two ways: either by altering the normal transcripts of a gene or by acting as an insulator. All other springer-induced alleles whose expression has been characterized to date produce aberrant transcripts. This newly discovered similarity between springer and gypsy prompted a search for Su(Hw)-binding sites in springer, since Su(Hw) mediates gypsy insulation. Springer has no Su(Hw)-binding sites, so it must be bound by a different insulating protein (Smith, 2001). Su(lyh) may encode the springer insulator protein. Su(lyh) dominantly suppresses the da^lyh insulation of both the cis-acting enhancer and the cis-acting negative regulator. The dominant nature of this suppression may be unique to the da locus, since autoregulation will amplify even the small amount of da gene product that results when the insulation is only weakly suppressed. However, su(Hw) can act as a dominant suppressor of some gypsy-induced alleles. Suppression of the da^lyh mutant phenotype is completely penetrant when the insulator protein is eliminated altogether, as in the case of Su(lyh) homozygotes. It is expected that the Su(lyh) protein will function like Su(Hw) protein by binding specific sites within springer (Smith, 2001).

In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. Evidence is provided that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic can not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye (Cadigan, 2002).

Overexpression of Wingless using P[sev-wg] results in flies lack interommatidial bristles, due to Wg repression of proneural gene expression. To utilize this phenotype as a starting point to identify genes that interact with wg, P[sev-wg^ts] flies were created that express a temperature-sensitive form of Wg. At 25°C, these animals have the normal (600/eye) number of bristles. At 16°C, where the Wg^ts protein is almost fully active, less than 50 bristles remain. At 17.6°C, approximately 150-200 bristles form. This temperature was chosen to generate a sensitized background with which to screen for dominant modifiers (Cadigan, 2002).

Focus was placed on three enhancers of the P[sev-wg^ts] bristle phenotype. All three reduce the number of bristles to between 10-50/eye. These modifiers form one lethal complementation group, which was meiotically mapped to an area between 30-32 on the cytological map. Complementation with deficiencies narrowed the region to 31B-32A, a location that includes the da gene. Four lines of evidence demonstrate that these enhancers are alleles of da: (1) they fail to complement lethal alleles of da and are rescued by a P[da⁺] rescue construct; (2) null alleles of da dominantly enhance the P[sev-wg^ts] phenotype; (3) clones of two modifiers were negative for Da antibody staining; (4) identical effects on proneural gene expression, bristle formation and MF progression were observed in clones of the modifiers and known alleles of da (Cadigan, 2002).

Mechanosensory bristles are four-cell external sensory organs that are derived from single sensory organ precursors (SOPs). In the wing and eye, SOP specification requires the proneural genes ac and sc, which encode bHLH transcription factors. These genes are first expressed in groups of cells known as proneural clusters. As one cell reaches the threshold of Ac/Sc expression necessary to trigger SOP formation, it represses proneural gene expression in the other cells in the cluster. This occurs through a process referred to as lateral inhibition, involving Notch signaling (Cadigan, 2002).

The Ac and Sc proteins are thought to promote SOP formation by acting with Da, another bHLH protein. da alleles dominantly suppress the ectopic bristle phenotypes caused by the misexpression of sc and lethal of scute (lsc), a gene that mimics ac/sc. Da can bind to Ac or Sc, and the heterodimers can bind to specific DNA sequences known as E boxes. In cultured cells, Da and Ac or Sc synergistically activate reporter genes with promoters containing E boxes, including the proximal promoter of the ac gene. Unlike Ac and Sc, all cells examined express some Da, though there is significant modulation of levels in some embryonic tissues and the eye. Because most of the spatial information is manifested in Ac and Sc, Da is thought of as a proneural gene co-factor (Cadigan, 2002).

To further examine the relationship between Da and Ac/Sc and bristle formation, clones of da were examined in the eye and wing. While large clones in the eye result in a total lack of eye development because of a block in MF progression, small clones differentiate eye tissue that completely lack interommatidial bristles. Ac expression is reduced at 3 hours after pupae formation (h APF) in da clones. This reduction of Ac protein and sc mRNA expression is also seen in the presumptive wing margin. Thus, da is required for normal proneural gene expression, most likely at the level of autoactivation, preventing the high expression levels needed for SOP specification (Cadigan, 2002).

At 3h APF, every cell in the basal portion of the eye (where Ac is expressed) expresses Da. However, groups of two to three cells have a much higher level of expression. Double staining with Ac indicates that these are the proneural clusters. In P[sev-wg] eyes, where Ac expression is greatly reduced, Da expression is also significantly lower. In P[GMR-Gal4], P[UAS-wg] (GMR/wg) eyes, ectopic wg is expressed at a much higher level than P[sev-wg]. GMR/wg eyes have almost no detectable Da or Ac expression (Cadigan, 2002).

Does the ability of ectopic wg to repress Da and Ac expression reflect a normal role for wg in bristle inhibition? Normal adult eyes lack interommatidial bristles at the periphery of the eye. An inverse correlation between Wg expression and that of Ac was found at the edge of early pupal eyes. However, clones removing wg activity are identical to wild type, with no extra bristles at the eye's periphery. Clones of arm, in contrast, almost always cause bristles to form right up to the edge of the adult eye. Since loss of Arm activity is normally associated with a block in Wg signaling, this result suggests that endogenous Wg signaling may repress bristle formation at the periphery (Cadigan, 2002).

The lack of ectopic bristles in wg clones could be explained by the fact that wg clones often act non cell-autonomously due to Wg diffusing in from surrounding wg⁺ tissue. However, temperature shifts (from 12 hours before pupation to 12 h APF) with wg^ts animals results in only occasional ectopic bristles. Clones of Df(2L)RF exhibit ectopic bristles one third of the time. This deficiency has reported breakpoints of 27F2-4-28A3. While it may delete up to 30 annotated genes besides wg (27F3), these include three other Wnt genes; Dwnt4 (27E7-27F1) Dwnt6 (27F3-5) and Dwnt10 (27F5-6). The removal of Dwnt4 was confirmed by in situ hybridization of Df(2L)RF homozygous embryos. Thus it is possible that one or more of these Wnts acts through arm to repress bristle formation at the edge of the eye (Cadigan, 2002).

Expression of wg at high levels behind the furrow (via the GMR promoter) results in a dramatically reduced eye completely lacking bristles. The reduced eye size is not due to a lack of MF progression. GMR/wg eyes have a large degree of apoptosis during pupal development that is partially responsible for the reduction in eye size. Coexpression of wg with a dominant negative form of TCF (pangolin-FlyBase), the transcription factor that mediates many Wg transcriptional effects, suppresses the size reduction of the GMR/wg eye and bristle inhibition. Ac and Da levels are also greatly elevated compared to GMR/wg/lacZ controls. These results, plus the requirement for arm shown for P[sev-wg] indicate a canonical Wnt pathway mediating these effects (Cadigan, 2002).

Since Wg signaling represses both Da and Ac expression, and Ac expression depends on da activity, it is possible that Wg represses Ac through inhibition of Da. One piece of evidence in support of this is that Da levels are only modestly affected in animals lacking ac and sc. Because lack of ac/sc does not cause lack of Da, the simplest model is that Ac is repressed by Wg signaling due to Da inhibiton (Cadigan, 2002).

To further test this hypothesis, attempts were made to rescue the Wg-induced bristleless phenotype by coexpression with either Da or Sc. If the simple model were correct, placing da under a heterologous (i.e., GMR) promoter would restore bristles to GMR/wg eyes, but expressing sc in the same way would not. If GMR/wg represses both da and sc directly (by direct, it is meant without influence of the other gene), then neither da or sc heterologous expression would restore bristles (Cadigan, 2002).

Surprisingly, the results do not follow either of the above models. Both da and sc coexpression rescue the bristleless phenotype of GMR/wg eyes. Not every bHLH protein can rescue the bristles; GMR/wg/ato eyes are still completely bristleless. The GMR/wg/da eyes have a significant but modest increase in Ac levels while GMR/wg/sc eyes show a similar degree of increase of Ac and Da expression. These results suggest a more complicated situation, though caution is needed when interpreting overexpression studies (Cadigan, 2002).

In addition to its role in SOP specification, da is also known to be required for the initiation and progression of the MF. Da is expressed at higher levels in the MF than elsewhere in the eye imaginal disc. It is thought to form heterodimers with the bHLH protein Atonal to specify R8 differentiation, which then promotes MF progression (Cadigan, 2002).

wg is known to be required for the proper orientiation of the MF. Removal of wg causes ectopic furrow initiation from the dorsal and ventral borders of the eye disc. In addition, ectopically expressed wg can block MF initiation and progression. Having established that Wg blocks bristle formation through (at least in large part) Da repression prompted an investigation of the possibility of a similar connection in MF initiation (Cadigan, 2002).

Misexpression of wg using a Dpp-Gal4 driver, which is active at the posterior edge of the eye disc, causes a complete block in MF initiation. Co-expression with the dominant negative TCF construct significantly rescues the block. Expression of an activated form of arm also blocks MF initiation, though not quite to the same extent as wg. As with bristle inhibition, wg appears to block MF initiation through a canonical Wnt pathway (Cadigan, 2002).

The strategy to test an involvement of da in wg-mediated MF inhibition is similar to that employed in the investigation of bristles. Coexpression of da with wg with Dpp-Gal4 always restores some MF progression. da also causes a dramatic increase in MF progression in Dpp/arm^act eyes. Similar to what was found for sc with bristle formation, expression of ato with wg results in a significant rescue of MF progression. The degree of rescue is less than that observed with da. Expression of sc with wg also gives a modest rescue of the MF, with 7 of the 12 Dpp/wg/sc eyes examined showing some MF progression. This result is surprising, since sc has no known physiological role in regulating the MF and highlights the potential pitfalls of overexpression studies (Cadigan, 2002).

The genes dpp and hh have also been implicated in MF initiation and progression. Coexpression of dpp with wg does not result in any rescue. Dpp-Gal4 driving P[UAS-wg] and P[UAS-hh] never results in MF rescue from the posterior edge. However, the majority of the eyes had at least one ectopic furrow that initiated from the anterior portion of the eye. These furrows apparently had different initiation times, since some were quite small, some had progressed to several rows of concentric photoreceptors and some had progressed to fill most of the eye disc. At least in regard to the initiation of the endogenous furrow at the posterior end of the eye, the results suggest that Wg acts at the level of da/ato, rather than hh or dpp (Cadigan, 2002).

Drosophila lilliputian is required for proneural gene expression in retinal development

Proper neurogenesis in the developing Drosophila retina requires the regulated expression of the basic helix-loop-helix (bHLH) proneural transcription factors Atonal (Ato) and Daughterless (Da). Factors that control the timing and spatial expression of these bHLH proneural genes in the retina are required for the proper formation and function of the adult eye and nervous system. This study reports that lilliputian (lilli), the Drosophila homolog of the FMR2/AF4 family of proteins, regulates the transcription of ato and da in the developing fly retina. lilli controls ato expression at multiple enhancer elements. lilli was found to contributes to ato auto-regulation in the morphogenetic furrow by first regulating the expression of da prior to ato. FMR2 regulates the ato and da homologs MATH5 and TCF12 in human cells, suggesting a conservation of this regulation from flies to humans. It is concluded that lilli is part of the genetic program that regulates the expression of proneural genes in the developing retina (Distefano, 2012).

This study has shown that lilli, the Drosophila homolog of the FMR2/AF4 family of proteins, regulates the transcription of the bHLH proneural genes atonal and daughterless in the developing retina and antenna of flies. It was further shown that this transcriptional regulation is conserved from flies to human cells. The data suggest that lilli regulates ato differentially at the 5' and 3' enhancer elements. The 3' cis-regulatory element is a 1.2-kb region of DNA located approximately 3.1 kb downstream of the ato transcription unit, and controls the early phase of ato expression in the developing retina. At this element, lilli appears to regulate transcription of ato at multiple sites (Distefano, 2012).

While analysis of transcription driven by a 5.8-kb element described previously is significantly reduced to less than 25% of controls, transcription driven by a minimal 348-bp enhancer element found within the larger 5.8-kb 3' enhancer is only reduced to 55% of controls. An important question remains for lilli-mediated ato transcription: is lilli function required to directly induce ato expression, or rather to maintain expression once previously induced? Given its function as a transcriptional activator, lilli function may be directly required to turn on ato expression at the 348-bp enhancer element in the developing retina. However, lilli may also be required to maintain ato expression once activated by other factors (such as Sine oculis or Eyeless/ Pax6) at this enhancer element. Alternately, other cis-regulatory elements along the 5.8- kb enhancer region may require lilli function to modulate ato expression after activation. Further experimentation will be required to answer these questions (Distefano, 2012).

The data also show that lilli-mediated regulation of ato expression at the 5' enhancer region requires the expression of the Da protein. The ato protein auto-regulates its expression at the 50 enhancer region to sustain ato gene expression in the intermediate groups and single R8 photoreceptors. It is hypothesized that the lilli protein regulates ato expression indirectly, by affecting ato auto-regulation at the 5' enhancer element. This hypothesis on multiple observations. (1) ato protein expression and ato transcription from the 5' enhancer element is decreased, but not absent within lilli mutant clones, consistent with an indirect effect on ato transcription. (2) Da protein expression and da transcription is also decreased, but not absent within lilli mutant clones in the region corresponding to the 5' enhancer expression. (3) By replacing Da expression within lilli mutant clones, ato transcription directed by the 5' enhancer element can be fully rescued, but not ato transcription directed by the 3' enhancer element. This is also true for ato protein expression in these clones (Distefano, 2012).

Thus, the lilli protein must first induce da transcription in cells in the intermediate groups and R8s prior to ato expression within these cells. Then, Ato-Da heterodimers can form, and maintain the activation of ato expression within the intermediate groups and R8 cells. If lilli function is compromised, da expression decreases, as does ato's ability to auto-regulate (Distefano, 2012).

This study has shown that lilli is required for the proper expression of hairy, another bHLH factor that is expressed anterior to the furrow. Hairy is an inhibitory factor to furrow progression. Interestingly, ato and da expression does not expand anteriorly in lilli clones, suggesting that loss of hairy anterior to the furrow is not sufficient to remove all of hairy function in these clones. Still, it is interesting that this analysis has identified a third bHLH factor regulated by lilli, and may suggest that lilli protein functions broadly to regulate the expression of multiple bHLH factors in different tissues (Distefano, 2012).

lilli is homologous to the AF4/FMR2 family of nuclear proteins in humans. This family includes FMR2, LAF4, AF4, and AF5Q31. Members of this family are implicated in acute lymphoblastic leukemia and FRAXE nonsyndromic Fragile X mental retardation (Distefano, 2012).

This study has shown that FMR2 also regulates the expression of the proneural genes MATH5 and TCF12 in HEK293 cells, showing that the observations made in the fly retina are conserved in human cell. Patients with FRAXE exhibit various developmental and morphological problems, including mental retardation, delays in speech development, attention deficit disorder, hyperactivity, and impaired motor coordination. While the etiology of these symptoms remains unknown, defects in neurogenesis, and/or the regulation of critical transcription factors such as the human homologues of ato and Da may be related to these symptoms. Further, recent evidence has shown that ato also functions as a tumor suppressor gene. This may provide an additional link between the misregulation of bHLH protein in lilli mutants and the leukemia observed in AF4 mutants. Further research is required to determine the significance of this connection (Distefano, 2012).

Targets of Activity

Daughterless is required for the activation of Sex lethal. In its absense, sxl is not activated and all progeny become males. Thus daughterless has an important role in sex determination (Crommiller, 1987).

sex lethal is regulated by helix-loop-helix (HLH) proteins such as Sisterless-b (Scute), Deadpan and Extramacrochaetae () . DA/SIS-B heterodimers bind several sites on the sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Repression by the DPN product of DA/SIS-B activation of sex-lethal results from specific binding of DPN protein to a unique site within the promoter. This contrasts with the mode of EMC repression, which inhibits the formation of the DA/SIS-B heterodimers. (Hoshijima, 1995).

Mutational loss of da does not affect Scute expression. However, loss of da reduces or outright eliminates the expression of five neuronal precursor genes: prospero, deadpan, asense, cyclin A and scratch. Similarly, there is little or no expression of the PNS-specific neuronal precursor gene couch potato in da mutants (Vaessin, 1994).

Daughterless has been found to regulate the expression of calmodulin (Kovalick, 1992) and deadpan (Younger-Shepherd, 1992) in the fly nervous system.

Proneural bHLH proteins Lethal of scute and Daughterless can both bind to and activate the promoters of the Enhancer of split complex and achaete. Daughterless binds to and activates sites within the first thousand upstream bases of the achaete transcription start site (van Doren, 1991). The target sequences called N boxes, differ slightly from the consensus E-box, usually bound by bHLH proteins. Two products of the E(spl)-C (hlh-m5 and Enhancer of split) attenuate the transcriptional activation mediated by proneural genes (Oellers, 1994). Extramachrochaete and Hairy serve this inhibition function as well (van Doren, 1991).

Dorsal and Daughterless interactions are required for the activation of twist and snail. Normally both genes are expressed in the ventral-most 18-20 cells of the embryo. In double dorsal/daughterless heterozygotes, the limits include just 10-12 cells. In addition there are gaps in expression near the cephalic furrow. This attests to the involvement of daughterless in dorsal-ventral polarity (Gonzalez-Crespo, 1993).

atonal expression has been analyzed in eye discs which have mutant daughterless clones. In morphogenetic furrow spanning mutant da clones, no or very little ATO portein expression is observed in apical confocal sections. The apical position is normal for photoreceptor cell nuclei at the time of differentiation. Basal focal sections of these same clones reveal that ATO is still present but is is abnormal in two respects: either it is shifted posteriorly or expressed in a wider than normal stripe. R8 expression of ATO within mutant da clones is also abolished. These results demonstrate that loss of da function does not affect the activation of striped ATO expression at the anterior side of the furrow. However, da may only be required for the expression of ATO within R8 cells. Alternatively, da may function in another cellular process within the furrow upon which photoreceptor cell determination depends. Although mutant ato does not affect the expression of early striped DA in the furrow, DA is not found in late third instar and early pupal mutant atonal eye discs (Brown, 1996).

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).

For Drosophila flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless proteins are known to form heterodimers that bind E-box sites and activate transcription. SISB-Da binding at Sxl was examined by using footprinting and gel mobility shift assays and SISB-Da was found to bind numerous clustered sites in the establishment promoter Sxl_Pe. Surprisingly, most SISB-Da sites at SxlPe differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. The noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for Sxl_Pe activity in embryos. Together with deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the Sxl_Pe switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements (Yang, 2001).

Deletion analysis has suggested that two subsegments within the 1.4-kb promoter account for most Sxl_Pe enhancer activity. An upstream segment (-1.4 to -0.8 kb) contributes to the strength of the promoter but is not essential for sex specificity. A proximal segment, including the start site and 390 upstream base pairs, drives a low-level, nonuniform female-specific expression. The sequence conservation between Sxl_Pe in D. melanogaster and Drosophila subobscura correlates well with the functional analysis. There is extensive sequence identity in the proximal region, with more limited matches in the distal segment, and no detectable similarity in the similarly sized central spacer segment. Within the proximal 390 bp, the sequences of all six B/Da binding sites are perfectly conserved. In the distal region, E-box sites 7 and 8 are conserved. Interestingly, while the sequence of site 9 is not conserved, another low-affinity B/Da site, CAGCTTG, is present in the equivalent position in D. subobscura (Yang, 2001 and references therein).

A critical question for sex determination is as follows: how can Sxl_Pe sense the twofold difference in male and female SIS and Runt concentrations and translate that into a strong all-or-nothing response? At some level, Sxl_Pe expression must be related to sex-specific differences in binding site occupancy. This is true whether dose sensitivity arises from cooperative DNA binding, competition with negative regulators, or from the sum of multiple independent interactions between the sex signal elements and the transcription machinery. It appears that sex-specific control of Sxl_Pe occurs largely through the activity of two regulatory regions: a central segment located between 1.4 and -0.8 kb, responsible primarily for promoter strength, and a proximal element, -390 to +44 bp, largely responsible for sex specificity. While these regions appear most important, sequences beyond -1.4 kb also contribute to the promoter, as inferred from the stronger lacZ expression from larger promoter fusions and by the ability of upstream sequences to partially substitute for the loss of the central -1.4 to -0.8 kb region (Yang, 2001).

The 10 B/Da sites identified in vitro are located in the central and proximal promoter elements. In addition, the sequence predicts 11 likely B/Da binding sites of high or moderate binding affinity located in the distal region between -1.6 and -3.7 kb, raising the possibility that there may be 21 or more B/Da sites in the functional Sxl_Pe region. Given a 39% GC content, random sequence would predict only 2.7 matches to the B/Da consensus at Sxl_Pe, suggesting that many of these predicted sites are functional binding sequences. Overall, there is a striking positional gradient of predicted binding affinities of the B/Da sites, with the moderate-affinity sites clustered proximally and the highest-affinity sites positioned distally. The asymmetric distribution of high- and moderate-affinity sites hints that the distal sites may be occupied at both high and low B/Da concentrations, with full occupancy of the proximal sites occurring only in XX embryos. This suggests a model in which the on or off response of Sxl_Pe to X-chromosome dose occurs primarily within the proximal X-counting region (XCR), with the distal segments providing an augmentation function that enhances transcription only when the female-specific XCR complex forms. It is unlikely that the distal high-affinity sites titrate B/Da from the XCR in males, because B/Da is in enormous excess over the Sxl binding sites (Yang, 2001).

Transcriptional control of stem cell maintenance in the Drosophila intestine

Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. This study found that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and genes of the Enhancer of split complex [E(spl)-C] were identified as the major targets of this repression. In addition, it was found that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. It is proposed that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation (Bardin, 2010).

Adult stem cells self-renew and, at the same time, give rise to progeny that eventually differentiate. This work provides evidence that one of the strategies used to maintain the identity of ISCs in Drosophila is to repress the expression of Notch target genes. Consistent with this finding, the loss of a general regulator of transcriptional repression, the Histone H2B ubiquitin protease Scrawny, gives a similar phenotype to Hairless (Buszczak, 2009). Additionally, several recent studies indicate that transcriptional repression of differentiation genes may be a central hallmark of stem cells in general (Bardin, 2010).

Two models have been proposed for Hairless activity. One proposes that Hairless competes with NICD for interaction with Su(H), thereby preventing transcriptional activation of Notch target genes by low-level Notch receptor activation. A second, non-exclusive, model proposes that Hairless antagonizes the transcriptional activation of Notch target genes by tissue-specific transcription factors other than Notch. Since the loss of Su(H) can suppress the phenotype of Hairless on ISC clone growth, it is proposed that Hairless promotes ISC maintenance by repressing the transcription of genes that would otherwise be activated by Notch signaling in ISCs. Thus, Hairless appears to set a threshold level to buffer Notch signaling in ISCs. In the absence of this repression, the expression of E(spl)-C genes and other Notch targets would lead to loss of the ISC fate (see Model for ISC maintenance). Importantly, these findings suggest a mechanism for how the transcriptionally repressed state is turned off and activation of the differentiation program is initiated: high activation of Notch in enteroblasts (EBs) displaces Hairless from Su(H) and leads to expression of the E(spl)-C genes (Bardin, 2010).

It is proposed that Hairless prevents ISC loss by repressing expression of Notch target genes, including the E(spl)-C genes. It is further proposed that Da-dependent bHLH activity promotes ISC identity, including the ability to self-renew and to express Delta. Delta, in turn, activates Notch in the adjacent EB, releasing the intracellular domain of Notch (NICD). It is speculated that, in response to Notch activation, the E(spl)-bHLH repressors downregulate Da-dependent bHLH activity in EBs as described in other systems, thereby shutting off ISC identity and promoting differentiation (Bardin, 2010).

E(spl)-C bHLH repressors act in part through their ability to inhibit bHLH activators. The data demonstrate that Da is also essential to maintain ISC fate and that E-box Da-binding sites are required to promote ISC-specific enhancer activity. Thus, it is proposed that activation of E(spl)-C genes by Notch in EBs downregulates Da bHLH activity and thereby contributes to turning off ISC identity in the differentiating cell (see Model for ISC maintenance). The specificity of ISC-specific E-box expression might be due to the ISC-specific expression of a bHLH family member. Although an array analysis raised the possibility that Scute may be specifically expressed in ISCs, genetic analysis indicates that scute function is not essential for ISC maintenance. Alternatively, specificity of gene expression might result from inhibition of bHLH activity in the EB and differentiating daughters, possibly by E(spl)-bHLH factors, rather than by the ISC-specific expression of a Da partner. It is also possible that a non-bHLH, ISC-specific factor restricts the Da-dependent bHLH activity to ISCs in a manner similar to the synergism observed in wing margin sensory organ precursors (SOPs) between the Zn-finger transcription factor Senseless and Da (Bardin, 2010).

Recently, a role for the Da homologs E2A (Tcf3) and HEB (Tcf12) has been found in mammalian ISCs marked by the expression of Lgr5 and, in this context, E2A and HEB are thought to heterodimerize with achaete-scute like 2 (Ascl2), which is essential for the maintenance and/or identity of Lgr5⁺ ISCs (van der Flier, 2009). In Drosophila, however, AS-C genes are not essential for ISC maintenance, but appear to play a role in enteroendocrine fate specification. The observation that Da bHLH activity is required for the identity of both Drosophila ISCs and mammalian Lgr5⁺ ISCs suggests that there might be conservation at the level of the gene expression program. Additionally, the bHLH genes Atoh1 (Math1) and Neurog3 are both important for differentiation of secretory cells in the mammalian intestine. Clearly, further analysis of the control of Da/E2A bHLH activity, as well as of the gene networks downstream of Da/E2A, will be of great interest (Bardin, 2010).

The data suggest that ISC fate is promoted both by inhibition of Notch target genes through Hairless/Su(H) repression and by activation of ISC-specific genes through bHLH activity. How then is asymmetry in Notch activity eventually established between the two ISC daughters to allow one cell to remain an ISC and one cell to differentiate? Three types of mechanisms can be envisioned that would allow for asymmetry of Notch signaling (Bardin, 2010).

First, the binary decision between the ISC and EB fates might result from a competition process akin to lateral inhibition for the selection of SOPs. In this process, feedback loops establish directionality by amplifying stochastic fluctuations in signaling between equivalent cells into a robust unidirectional signal. The finding that the Da activator and E(spl)-bHLH repressors are important to properly resolve ISC/EB fate is consistent with this type of model. Activation of the Notch pathway in one of the daughter cells may then lead to the changes in nuclear position (Bardin, 2010).

Second, the asymmetric segregation of determinants could bias Notch-mediated cell fate decisions. The cell fate determinants Numb and Neur are asymmetrically segregated in neural progenitor cells to control Notch signaling. However, no evidence was found for the asymmetric segregation of these proteins in dividing ISCs. Additionally, the data indicate that Numb is not important to maintain ISC fate. It cannot be excluded, however, that another, unknown Notch regulator is asymmetrically segregated to regulate the fate of the two ISC daughters (Bardin, 2010).

A third possibility is that after ISC division, one of the two daughter cells receives a signal that promotes differential regulation of Notch. Indeed, it has been noted that the axis of ISC division is tilted relative to the basement membrane, resulting in one of the progeny maintaining greater basal contact than the other. An extracellular signal coming either basally or apically could bias the Notch-mediated ISC versus EB fate decision. For instance, Wg secreted by muscle cells could act as a basal signal to counteract Notch receptor signaling activity in presumptive ISCs. This could be accomplished by Wg promoting bHLH activity or gene expression. Indeed, Wg has been demonstrated to promote proneural bHLH activity in Drosophila (Bardin, 2010 and references therein).

These models are not mutually exclusive, however, and proper control of ISC and differentiated cell fates during tissue homeostasis might involve multiple mechanisms (Bardin, 2010).

A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells

Neurogenesis is initiated by a set of basic Helix-Loop-Helix (bHLH) transcription factors that specify neural progenitors and allow them to generate neurons in multiple rounds of asymmetric cell division. The Drosophila Daughterless (Da) protein and its mammalian counterparts (E12/E47) act as heterodimerization factors for proneural genes and are therefore critically required for neurogenesis. This study demonstrates that Da can also be an inhibitor of the neural progenitor fate whose absence leads to stem cell overproliferation and tumor formation. This paradox can be explained by demonstrating that Da induces the differentiation factor Prospero (Pros) whose asymmetric segregation is essential for differentiation in one of the two daughter cells. Da co-operates with the bHLH transcription factor Asense, whereas the other proneural genes are dispensible. After mitosis, Pros terminates Asense expression in one of the two daughter cells. In da mutants, pros is not expressed, leading to the formation of lethal transplantable brain tumors. These results define a transcriptional feedback loop that regulates the balance between self-renewal and differentiation in Drosophila optic lobe neuroblasts. They indicate that initiation of a neural differentiation program in stem cells is essential to prevent tumorigenesis (Yasugi, 2014).

To further characterize the overproliferation caused by da RNAi, da RNAi was induced by insc-Gal4 in all larval NBs. The number of Deadpan (Dpn) expressing NBs increased at the expense of Embryonic lethal abnormal vision (Elav) expressing neurons. Although da was expressed in all NBs of the central brain and in some progenitor cells, no phenotype was found in these lineages when da³ amorphic mutant clones were induced using mosaic analysis with a repressible cell marker (MARCM) technique (Yasugi, 2014).

The visual processing centers of the fly brain consist of the so-called optic lobes. The medial surface of the optic lobes is surrounded by medulla NBs that differentiate from NE cells and generate medulla neurons on the inner side of the brain. In the optic lobe, da is expressed in NE cells and in medulla NBs. To induce daRNAiin the optic lobe, a dpn-Gal4 driver line was used that showed strong Gal4 expression in NE cells and medulla NBs and weak expression in medulla neurons (called dpnOL-Gal4). Expression of daRNAi using dpnOL-Gal4 caused a strong increase of Dpn positive NBs. The da RNAi phenotype was examined with the mitotic marker Phospho-Histone H3 (PH3), the NB marker Miranda (Mira) and the neuronal marker Elav. In the wild type, PH3 positive mitotic cells (NBs and GMCs) were restricted to the periphery of the optic lobe. In da RNAi samples, PH3 positive cells were mislocalized and ectopically found in the inner side of the brain. To confirm this phenotype, da³ mutant clones were induced in the optic lobe. In da³clones, Dpn positive NBs were found in the region that was normally occupied by medulla neurons. Thus, da is required for cell fate determination in medulla NBs (Yasugi, 2014).

To test whether the ectopic NBs in da RNAi brains have unlimited growth potential and can induce malignant tumors, optic lobes expressing GFP under the control of insc-Gal4, were dissected and implanted into the abdomen of wild type adult host flies. Transplanted cells from da RNAi brains proliferated and GFP positive cells were observed in the host flies, while no substantial growth was observed in control samples. PH3 positive mitotically active cells were observed in the tissue from transplanted daRNAi samples, and this tumor tissue consisted of both Dpn-expressing NB-like cells and Elav-expressing neuron-like cells. This suggests that the da tumor cells proliferate and some of the cells keep the stem cell state, but these cells also produce differentiating cells. This is consistent with the result from da³ clones, in which both ectopic NBs and differentiated neurons were observed. From these results, it is concluded that da acts as a tumor suppressor in optic lobe NB lineages (Yasugi, 2014).

Da is an E-box protein that heterodimerizes with other bHLH type transcription factors, such as the proneural proteins of the AS-C. The AS-C is composed of four transcription factors called Achaete (Ac), Scute (Sc), Lethal of Scute (L(1)sc), and Asense (Ase). While Ac is not expressed in the optic lobe, three of four AS-C proteins show specific expression. Sc is expressed in the NE cells and NBs, L(1)sc is transiently expressed in the transition zone between NE cells and NBs, and Ase is expressed in NBs and GMCs in the developing medulla. To test which of the AS-C genes might act with da during cell fate determination in medulla NBs, clones of several deletion lines were induced that uncover the AS-C region. Ectopic NBs were observed in clones of Df(1)260-1 uncovering all AS-C genes or in ase¹ that uncovers the ase coding region. On the other hand, no phenotype was observed in clones of Df(1)sc19, which deletes ac, sc, and l(1)sc. Since the phenotype of Df(1)260-1 or ase¹ clones was similar to the phenotype of da³ mutant clones and heterodimerization between Ase and da has been shown, it is concluded that da acts together with Ase to regulate cell fates in the optic lobe. It has been reported that da is required for the timely differentiation from NE cells to NBs and L(1)sc is involved in this transition during the optic lobe development. From the expression pattern of AS-C genes and results from the clonal analysis using deficiency lines, a dual function is proposed for Da: As a heterodimer with L(1)sc, da promotes the transition from NE cells to NBs. Later, da acts with Ase in NBs to promote differentiation and prevent tumor formation (Yasugi, 2014).

To identify the downstream targets of da and Ase, the expression was tested of candidate genes. The homeodomain transcription factor Pros acts as a cell fate determinant in embryonic and larval NBs and is regulated by da and Ase in embryos. In the larval optic lobe, Pros is localized to the basal cortex of dividing NBs and nuclear in GMCs and newly born medulla neurons. Whether Pros expression is dependent on da and/or Ase was tested. Pros expression decreased in da³ or ase¹ mutant clones suggesting that Pros acts downstream of da and Ase. To test whether pros is required for cell fate determination in the optic lobe, induced pros¹⁷ mutant clones were induced. In pros¹⁷ mutant clones, ectopic NBs were observed in the medulla neuron layer, which was similar to the phenotype of da³ or ase¹mutant clones. Overexpression of Pros, on the other hand, resulted in a decrease of medulla NBs. To test whether Pros acts downstream of Da, Pros was overexpressed in a da RNAi background. A reduced number of medulla NBs were observed in optic lobes overexpressing Pros in a da RNAi background, indicating that pros is epistatic to da. Thus, Pros is a key downstream target of da and Ase in optic lobe NBs (Yasugi, 2014).

Next, it was asked whether Pros expression is regulated by da in the central brain where da is not required for NB self-renewal. Nuclear Pros expression was found in differentiating daughter cells in the wild type. Pros expression remained in da³ mutant clones. Thus, unlike in the optic lobe, da is not essential for Pros expression in the central brain. This explains why the da phenotype is specific to the optic lobe NBs, while pros mutations cause overproliferation in all larval NBs. It is speculated that other factors may act redundantly to regulate Pros expression in the central brain (Yasugi, 2014).

If Pros is induced by da and Ase, then how are their functions turned off after asymmetric division? To test whether Pros can terminate the expression of ase, Ase expression was examined in pros¹⁷ clones. While Ase expression was restricted to the periphery of the optic lobe in wild type, Ase expression continued on the inner side of the optic lobe in pros¹⁷ clones. Thus, Pros turns off Ase expression and this transcriptional negative feedback loop regulates the proliferation and differentiation of NBs (Yasugi, 2014).

A prevailing view in stem cell biology is that a self-renewal program allows prolonged proliferation in stem cells and is turned off upon differentiation. The current data challenge this view and demonstrate that the ability to differentiate is pre-programmed in neural stem cells. This explains why transcription factors like da and Ase that are thought to be required for NB specification can be required for proper differentiation and act as tumor suppressors. It is proposed that a regulatory transcriptional loop assures cell fate determination and inhibits tumor formation. In a medulla NB, Da and Ase heterodimers induce Pros expression but Pros is excluded from the nucleus and therefore can not terminate Ase expression. After asymmetric cell division, however, Pros enters the nucleus of the GMC where it initiates differentiation and cell cycle exit. In the GMC, Pros terminates Ase expression and therefore triggers an irreversible decision towards differentiation. The data from embryonic NBs suggest that Pros can directly bind to the ase region and regulates its expression. In the absence of this regulation, GMCs maintain the stem cell fate and continue to grow into malignant tumors (Yasugi, 2014).

The role of Da, Ase, and Pros in neural stem cells could be conserved in mammals. Mammalian class I bHLH genes, namely E2A (encoding the E12 and E47 proteins), E2-2, and HEB are expressed in the developing brain. E2A, HEB, or E2A/HEB transheterozygous mutant mice show a brain size defect, suggesting that class I factors also regulate mouse brain development. Mash1 and Prox1, the vertebrate orthologs of Ase and Pros, are expressed in proliferating neural precursor cells of the developing forebrain and spinal cord. Like in Drosophila, Mash1 induces Prox1 and Mash1 promotes an early step of differentiation in neural stem cells. Like in vertebrates, NE cells in the Drosophila optic lobe first proliferate by symmetric cell division and then become asymmetrically dividing NBs. From these molecular and developmental similarities, it is speculated that the transcriptional regulatory mechanism this study identified might be well conserved in mammalian brains (Yasugi, 2014).

The data are of particular relevance in light of the recently postulated role of stem cells in the formation of malignant tumors. Failure to limit self-renewal capacity in stem cells or defects in progenitor cell differentiation can both lead to the formation of cells that continue to proliferate and ultimately form tumors. While genes acting in stem cells are thought to promote self-renewal, genes required in differentiating cells are thought to promote differentiation and limit proliferation and are therefore candidate tumor suppressors. The current data challenge this view and show that the path to differentiation is initiated in the stem cell and therefore even genes specific to stem cells can act as tumor suppressors. It will be interesting to determine whether a similar mechanism acts in mammalian neural stem cells as well. If it does, the expression pattern of a gene can no longer be used as a main criterion for whether it promotes or inhibits self-renewal in stem cell lineages (Yasugi, 2014).

Salvador-Warts-Hippo pathway in a developmental checkpoint monitoring Helix-Loop-Helix proteins

The E proteins and Id proteins are, respectively, the positive and negative heterodimer partners for the basic-helix-loop-helix protein family and as such contribute to a remarkably large number of cell-fate decisions. E proteins and Id proteins also function to inhibit or promote cell proliferation and cancer. Using a genetic modifier screen in Drosophila, this study shows that the Id protein Extramacrochaetae enables growth by suppressing activation of the Salvador-Warts-Hippo (SWH) pathway of tumor suppressors, activation that requires transcriptional activation of the expanded gene by the E protein Daughterless. Daughterless protein bound to an intronic enhancer in the expanded gene, both activated the SWH pathway independently of the transmembrane protein Crumbs and bypassed the negative feedback regulation that targets the same expanded enhancer. Thus, the Salvador-Warts-Hippo pathway has a cell-autonomous function to prevent inappropriate differentiation due to transcription factor imbalance and monitors the intrinsic developmental status of progenitor cells, distinct from any responses to cell-cell interactions (Wang, 2015).

This study describes a process that prevents certain misspecified cells from differentiating into malformed organs. This process creates a requirement for the emc gene in imaginal disc cell growth, since emc loss results in high Da levels that trigger the pathway through transcriptional activation of the ex gene, an upstream regulator of the SWH tumor suppressor pathway. If ex or the downstream SWH genes are mutated, then cells with high Da levels not only survive and grow but also produce numerous ectopic neuronal structures. This surveillance function for SWH signaling does not require cell-cell signaling and is distinct from potential roles for SWH in limiting organ growth or preventing tumorigenesis. It may represent an adaptive function for SWH pathway hyperactivity (Wang, 2015).

The heterodimer partners of Da and Emc include proneural bHLH proteins that define proneural regions and neural progenitor cells and that are highly regulated in space and time. Da, by contrast, is expressed ubiquitously and controlled by emc. Inadequate emc expression permits higher levels of da expression and Da/bHLH heterodimers, leading to ectopic neural differentiation. Mammalian Id genes are similar feedback regulators of mammalian E-proteins. This study has shown that even if emc expression or its regulation is defective, abnormal neurogenesis is still restrained by SWH signaling that restricts the proliferation and survival of cells with abnormal Da expression. High Da levels directly activate transcription of the ex gene, thereby activating the SWH pathway of tumor suppressors in a cell-autonomous fashion. Because ex is a feedback inhibitor of SWH signaling that is transcriptionally activated by Yki, ex activation by high Da has the added effect of bypassing feedback control of SWH signaling, which likely contributes to the efficiency of removal of cells with high Da. Indeed, when ex is removed, cells with high Da are not removed but produce dramatic neural hyperplasia, in which ectopic bristles almost cover a clone in the thoracic epidermis. All these neurogenic defects would be maladaptive in nature, where the pattern of sensory bristles is highly selected (Wang, 2015).

These findings suggest that the Da/Emc balance is permissive for normal growth, and no evidence was found for regulation that determines normal organ size or growth rate. By contrast, Da/Emc imbalance outside the normal range in mutant cells triggers the SWH pathway to block growth and remove cells that will otherwise perturb developmental patterning. SWH activation in abnormal development might be analogous to the p53 tumor suppressor, which is inactive in most normal cells, but activated by DNA damage and other stresses. Interestingly a recent study reported that emc hypomorphic cells, which are less severely affected that emc null cells and can survive in imaginal discs, nevertheless exhibit a growth deficit caused by repression of the cell cycle gene string/cdc25, and that string/cdc25 is repressed directly by abnormally high Da. Thus there may be multiple, Da-dependent pathways that converge to select against progenitor cells with incorrect cell-fate specification (Wang, 2015).

Mammalian E-proteins and Id-proteins are well-established tumor suppressors and proto-. In normal development, E proteins and Id-proteins regulate the coordination of differentiation with cell-cycle arrest and the expansion of mammary epithelial cells in response to pregnancy and lactation. At least in part, these growth controls relate to the transcriptional activation of cyclin-dependent kinase inhibitor genes by E-proteins, such that E-proteins are required for cellular senescence, counteracted by Id-proteins. The senescence mechanisms may not be conserved between mammalian and Drosophila cells, but other pathways of tumor suppression by mammalian E-proteins exist, and in certain contexts, E-proteins can be tumor promoting and Id-proteins tumor suppressive (Wang, 2015).

The distinctive phenotype of SWH pathway mutations is dramatically enhanced growth and organ size. The normal biological functions of the pathway are still debated. Reduced SWH activity is implicated in wound healing and regenerative growth. Mice mutant for Mst1, Mst2, Lats1, or Lats2 are tumor prone, suggesting that tumor growth could mimic wound healing or regeneration. Epigenetic silencing of these genes has been reported in human cancer, where other SWH components are mutated, such as NF2 in neurofibromatosis. Yap is amplified in cancers of the liver, colon, lung, and ovary (Wang, 2015).

Clearly, SWH activity is normally maintained between a low threshold necessary to prevent hyperplasia and a high threshold that blocks growth and kills cells. Reduced SWH activity is associated with regenerative responses. In principle, increased SWH might be hyperactivated to eliminate potential tumors, perhaps because of imbalanced expression of E-proteins and Id-proteins; tumor cells might evolve to evade such a checkpoint. Microarray data from E2A-deficiency mice that exhibit high incidence of T cell leukemia suggest that FRMD6, a mammalian homolog of ex, is an E2A target, which would be consistent with this hypothesis (Wang, 2015).

This work shows directly that in Drosophila hyperactivation of the SWH tumor suppressor pathway can select against cells that express certain developmental errors, which may be adaptive for development. It will be interesting to discover whether SWH signaling can be hyperactivated to remove other kinds of dysfunctional cells besides those expressing inappropriate bHLH protein levels, whether in development or in cancer (Wang, 2015).

Protein Interactions

Various bHLH proteins can bind as heterodimers to the E box consensus sequence G/ACAGNTGN. Daughterless binds as a heterodimer with achaete-scute complex genes. This interaction is antagonized by Extramachrochaete (van Doren, 1991). Atonal, when involved in the determination of chordotonal neurons also dimerizes with Daughterless (Jarman, 1993).

Classical genetics indicates that the achaete-scute gene complex (AS-C) of Drosophila promotes development of neural progenitor cells. To further analyze the function of proneural genes, the effects of Gal4-mediated expression of lethal of scute, a member of the AS-C, were studied during embryogenesis. Expression of lethal of scute forces progenitor cells of larval internal sensory organs to take on features of external sensory organs. Normally, these cells are committed to this fate independent of AS-C activity. Surprisingly, overexpression of l'sc does not result in supernumerary neural cells. Supernumerary neural cells can be induced ectopically only if daughterless is overexpressed, either alone or together with lethal of scute: cells of the amnioserosa and the hindgut then express neuronal markers. Cells of the proctodeal anlage, which normally lack neural competence, acquire the ability to develop as neuroblasts following transplantation into the neuroectoderm. Activated Notch prevents the cells of the neuroectoderm from forming extra neural tissue when they express an excess of proneural proteins. Under the present conditions, lateral inhibition is thus dominant over the activity of proneural genes (Giebel, 1997).

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and is antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. Since all these proteins bear a helix-loop-helix (HLH) dimerization domain, their potential pairwise interactions were investigated using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Daughterless, Achaete, Scute, and Extramacrochaetae. The seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. A subset of E(spl) proteins (MB and M5) can heterodimerize with Da, another subset (M3) can heterodimerize with proneural proteins, and yet another (Mbeta, Mgamma and M7) with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. An investigation was carried out of the structural requirements for some of these interactions, using site-specific and deletion mutagenesis. Deletion analysis of M3 and Scute is consistent with their interaction being mediated by their respective bHLH domains. The dependence of the E(spl)-activator HLH interactions on the HLH domain is nicely reflected in the fact that the functional grouping of the E(spl) proteins correlates well with the amino acid sequences of their bHLH domains, e.g., M5 and M8 have highly similar bHLH regions, different from those of the M7/Mbeta/ and Mgamma group, which also display high intragroup similarity. The strong interactions observed between E(spl) proteins and proneural proteins might lead one to hypothesize the E(spl) proteins act like Extramachrochaetae, i.e., by sequestering HLH activators. This is unlikely, since residual activities of E(spl) proteins with mutated basic domains have only weak residual activities (Alifragis, 1997).

The Dorsal morphogen has been implicated in the establishment of the embryonic mesoderm, neuroectoderm, and dorsal ectoderm. The simultaneous reduction in the levels of DL and any one of several helix-loop-helix (HLH) proteins results in severe disruptions in the formation of mesoderm and neuroectoderm. HLH proteins that have been implicated in neurogenesis (Daughterless, Achaete, and Scute) are required for the formation of these embryonic tissues. DL-HLH interactions involve the direct physical association of these proteins in solution mediated by the rel domain of Dorsal and HLH domains of the other transcription factors (Gonzalez-Crespo, 1993).

The ability of the absent MD neurons and olfactory sensilla (Amos) and Da proteins to form complexes in the presence of E boxes has been examined. Since the DNA-binding domain of Amos is almost identical to that of Ato, E box-containing oligonucleotides, E1 and E4, which represent high-affinity binding sites for the Da/Ato protein complex, were examined. The Sc/Da complex was used as a positive control since this complex also binds to these two E boxes. E1 and E4 boxes are well bound by Amos/Da. In contrast, this shifted complex including Amos and Da is undetectable with either Amos or Da alone. The Amos/Da complex is supershifted by addition of anti-GST antibody, which recognizes the GST-Da protein used in this assay. This binding of the Amos/Da complex to E boxes is efficiently completed by addition of excess E1 and E4 cold probes but not by two corresponding mutant E boxes. These data suggest that Amos and Da form a heterodimer when bound to E boxes and that the binding activities are sequence specific. To further analyze the interaction between amos and da in vivo, the effects of amos misexpression were examined in different da genetic backgrounds. The number of neurons were counted in sca-GAL4/UAS-amos embryos carrying different da gene dosages. When a moderate level of amos is induced in wild-type embryos with two copies of da⁺, some ectopic Elav-positive cells are observed. The ectopic neurons are suppressed in embryos carrying only one copy of da⁺. When amos and da are simultaneously misexpressed, numerous Elav-positive cells are induced. The strong neuralization by amos and da has also been revealed by the staining of MAb 22C10, which labels the neuronal morphology. These ectopic neurons include MD neurons that express lacZ from E7-2-36 insertion. As a control, misexpression of da causes only a minor effect on the number of neurons in this assay. These results suggest that the ectopic neuron formation elicited by amos is very sensitive to the gene dosage of da (Huang, 2000).

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip acts as a cofactor of the dorsal selector Apterous, and both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax (Ramain, 2000).

Pnr is a member of the GATA-1 family of transcription factors and activates proneural function by binding to the dorsocentral (DC) enhancer located 4 kb and 30 kb upstream of ac and sc, respectively. Reported in this study is the characterization of Chip^E, a viable allele of Chip, that interacts with pnr genetically. Chip^E mutants show reduced ac-sc expression in the DC, associated with loss of DC bristles, and produce a phenotype similar to that of loss-of-function pnr alleles. This genetic interaction correlates with a physical interaction between Chip, Pnr, and the bHLH heterodimers (Ac/Sc-Da). Pnr interacts with the N terminus of Chip through its COOH terminus encompassing two helices that are conserved between D. melanogaster and D. virilis and that are probably both involved in protein-protein interactions. Chip dimerizes with the bHLH heterodimers through its C-terminal LID, known to mediate heterodimerization with LIM-containing proteins (Ramain, 2000).

In vertebrates, the Ldb1/NLI protein associates with GATA-1, Lmo2, and the bHLH E47, Tal1/SCL in an erythroid complex whose function is poorly understood. A similar Drosophila complex functions in vivo to regulate the ac/sc genes directly during establishment of the proneural prepattern.

Accurate coexpression of ac/sc is mediated by Pnr binding to the DC. The ac and sc promoters include E boxes that are targets for the Ac/Sc-Da heterodimers and support autoregulation during development. In cultured chicken embryonic fibroblast (CEF) cells, Pnr and the Ac/Sc-Da heterodimer activate expression of a CAT reporter linked respectively to the DC enhancer and to the ac promoter. Physical interactions between Pnr and the bHLHs lead to synergistic activation of the reporter when the regulatory sequences are associated. Pnr and the Ac/Sc-Da heterodimers are both required in flies for expression of a LacZ reporter linked to the promoter associated with the enhancer, but the analysis of Chip^E shows that Chip is also required for full activation in vivo. The interactions between Pnr and the bHLH mediated by Chip suggest that Pnr might also be involved in autoregulation. Interestingly, Chip interacts with Ac/Sc through the Ac/Sc bHLH domains, and it has been shown that the overexpression of a homologous bHLH domain is sufficient to mediate the proneural function of Ac/Sc (Ramain, 2000).

Chip has been identified in a genetic screen for mutations that reduce activity of the wing margin enhancer of the cut locus, and it has been proposed that Chip may act as a bridge allowing enhancer-promoter communications. Thus, if the flies have a unique functional Chip allele, they display a cut margin phenotype, and this effect is observed exclusively when they carry a gypsy insertion between the enhancer and the promoter on one chromosome. It has been proposed that binding of the Su(Hw) insulator protein to the gypsy insertion blocks communication on the mutant chromosome, thereby interfering with the functioning of the wild-type homolog. The interchromosomal insulation is detectable when Chip activity is reduced, and Chip and Su(Hw) are antagonistic to each other, suggesting that Chip may be a facilitator target of Su(Hw) (Ramain, 2000).

The Chip^E mutation specifically disrupts interactions with the bHLH and strongly affects the expression of a LacZ reporter linked to the ac promoter/DC enhancer in flies, suggesting that Chip also mediates enhancer-promoter communication in the ASC. Further evidence is provided by the Hw¹ mutant. Hw¹ carries a gypsy insertion within ac such that sc, which is located further downstream from the DC enhancer, is no longer expressed. In addition, the removal of the gypsy insulator largely restores sc expression in the DC proneural cluster (Ramain, 2000).

Thus, a complex containing Pnr, Chip, and the Ac/Sc-Da heterodimer activates ac-sc expression, and its function is antagonized by Ush, Ap, and Emc. Ush and Emc dimerize respectively with Pnr and the HLH Ac/Sc. The repressing effect of Ap may reflect its ability to interact with Chip, thereby depriving Pnr of its essential cofactor. Alternatively, Ap may weaken the enhancer activity of Pnr. Thus, Ap may compete directly with Chip for binding to Pnr (Ramain, 2000).

Within the domain of Pnr expression, Ap and Pnr compete for binding to their common Chip cofactor. Ap activity is mediated by a Chip dimer, whereas activation of ac-sc by Pnr requires a Chip monomer. Chip acts as a bridge between the Ac-Da heterodimer bound to the E boxes of the ac promoter and Pnr bound to the GATA sites of the DC enhancer. The activity of the resulting complex is antagonized by Ush and Emc, which negatively regulate Pnr and Ac/Sc functions during development. The repressing effect of Ap is mediated either by dimerization of Ap with Chip and/or Pnr or by Chip-assisted binding of Ap to sites located between the DC enhancer and the ac promoter. Additional cofactors, such as dLMO, may participate in this complex (Ramain, 2000).

Chip is required in flies for ASC activation, whereas it appears dispensable in CEF cells. This observation may reflect the nature of the reporter used in the transfection experiments where the DC enhancer is close to the ac promoter and poorly mimics the genomic organization of the ASC, where the DC enhancer has to regulate ac and sc simultaneously. Furthermore, the chromatin structure and its modifications associated with gene expression are probably not reproduced in the transient expression assay. Thus, ASC expression in flies may require additional coactivators recruited by Chip, including chromatin remodeling factors. Moreover, the activation of ac/sc probably requires the assembly of a higher-order nucleoprotein complex containing multiple transcription factors (enhanceosome), and Chip may allow the correct positioning of Pnr and the Ac/Sc-Da heterodimer in this structure (Ramain, 2000).

Chip^E mutants affect the scutellar and dorsocentral bristles in opposite fashions. It will be of interest to compare the regulation of the activity of the corresponding enhancers by Chip and Pnr (Ramain, 2000).

It has been proposed that appropriate combinations of proteins represent the positional cues that activate a given enhancer of the ASC complex. The disc is divided in large territories, but almost nothing is known concerning how these territories are further subdivided or how the positional information revealed by the accurate ac-sc expression is created. The present study provides a link between the spatial regulation of the proneural genes and the compartmentalization of the disc. ac-sc expression is stimulated by a complex containing the prepattern factor Pnr, Chip, and the bHLH proteins Ac/Sc and Da. Chip is an essential cofactor of the dorsal selector Ap, and these interactions coordinate the spatial transcription of the proneural genes. Ap is expressed specifically in the dorsal compartment of the wing pouch, and the juxtaposition of Ap-expressing with Ap-nonexpressing cells defines the dorsal/ventral organizing boundary where wingless (wg) expression is induced. On the thorax, ap and Chip are ubiquitously expressed, whereas wg expression occurs in a stripe straddling the lateral border of the domain of pnr expression. Moreover, Pnr activates wg. Pnr associates with Chip, and the domain of pnr expression appears devoid of Ap activity. As a consequence, this domain may define a boundary between a region devoid of Ap activity and a region where Ap is active. Alternatively, Pnr may associate with Ap, and the resulting heterodimer may regulate wg. Further studies will help to resolve this issue (Ramain, 2000).

Schneider SL2 cells activate the myogenic program in response to the ectopic expression of daughterless alone, as indicated by exit from the cell cycle, syncytia formation, and the presence of muscle myosin fibrils. Myogenic conversion can be potentiated by the coexpression of Drosophila Mef2 and nautilus with daughterless. In RT-PCR assays Schneider cells express two mesodermal markers, Nautilus and Mef2 mRNAs, as well as very low levels of Daughterless mRNA but no Twist. Full-length RT-PCR products for Nautilus and Mef2 encode immunoprecipitable proteins. RNA-i was used to demonstrate that both endogenous nautilus expression and Mef2 expression are required for the myogenic conversion of Schneider cells by daughterless. Coexpression of twist blocks conversion by daughterless but twist dsRNA has no effect. These results indicate that Schneider cells are of mesodermal origin and that myogenic conversion with ectopic expression of daughterless occurs by raising the levels of Daughterless protein sufficiently to allow the formation of Nautilus/Daughterless heterodimers. The effectiveness of RNA-i is dependent upon protein half-life. Genes encoding proteins with relatively short half-lives (10 h), such as Nautilus or Hsf, are efficiently silenced, whereas more stable proteins, such as cytoplasmic actin or beta-galactosidase, are less amenable to the application of RNA-i. These results support the conclusion that Nautilus is a myogenic factor in Drosophila tissue culture cells with a functional role similar to that of vertebrate MyoD. This is discussed with regard to the in vivo functions of Nautilus (Wei, 2000).

Sage, a salivary-specific bHLH protein, has been identified as a new heterodimeric partner for Daughterless protein in the salivary glands

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).

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, 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).

A DNA transcription code for cell-specific gene activation by notch signaling

Cell-specific gene regulation is often controlled by specific combinations of DNA binding sites in target enhancers or promoters. A key question is whether these sites are randomly arranged or if there is an organizational pattern or 'architecture' within such regulatory modules. During Notch signaling in Drosophila proneural clusters, cell-specific activation of certain Notch target genes is known to require transcriptional synergy between the Notch intracellular domain (NICD) complexed with CSL proteins bound to 'S' DNA sites and proneural bHLH activator proteins bound to nearby 'A' DNA sites. Previous studies have implied that arbitrary combinations of S and A DNA binding sites (an 'S+A' transcription code) can mediate the Notch-proneural transcriptional synergy. By contrast, this study shows that the Notch-proneural transcriptional synergy critically requires a particular DNA site architecture ('SPS'), which consists of a pair of specifically-oriented S binding sites. Native and synthetic promoter analysis shows that the SPS architecture in combination with proneural A sites creates a minimal DNA regulatory code, 'SPS+A', that is both sufficient and critical for mediating the Notch-proneural synergy. Transgenic Drosophila analysis confirms the SPS orientation requirement during Notch signaling in proneural clusters. Evidence that CSL interacts directly with the proneural Daughterless protein, thus providing a molecular mechanism for this synergy. It is concluded that the SPS architecture functions to mediate or enable the Notch-proneural transcriptional synergy which drives Notch target gene activation in specific cells. Thus, SPS+A is an architectural DNA transcription code that programs a cell-specific pattern of gene expression (Cave, 2005).

The functional significance of the SPS element has not been determined, but initially, it was proposed that the arrangement of the S binding sites in the SPS may function to mediate cooperative DNA binding by CSL proteins, or it may be necessary for the recruitment of other proteins to the promoter. Subsequent studies, though, showed that CSL, NICD, and Mam "ternary complexes" can assemble on single S sites. To date, no studies have experimentally addressed whether there are significant functional differences between SPS elements and single S or other non-SPS binding site configurations, and the mechanistic function of the SPS element is not known (Cave, 2005).

In Drosophila, five of the seven bHLH repressor genes in the E(spl)-Complex contain an SPS element in their promoter regions, and four of these bHLH R genes contain both SPS and proneural bHLH A protein binding (A) sites. These four bHLH R genes (the m7, m8, mγ, and mδ genes, collectively referred to as the 'SPS+A bHLH R' genes have been shown genetically to depend upon proneural bHLH A genes for expression. In addition, transcription assays in Drosophila cells with at least two of these four genes (m8 and mγ) have shown that there is strong transcriptional synergy when NICD and proneural proteins are expressed in combination. These SPS+A bHLH R genes also have similar patterns of cell-specific expression within proneural clusters. Following determination of the neural precursor cell from within a proneural cluster of cells, Notch-mediated lateral inhibition is initiated and these SPS+A bHLH R genes are specifically upregulated in all of the nonprecursor cells but not in the precursor cell. The absence of NICD, and the presence of specific repressor proteins such as Senseless, prevent upregulation of SPS+A bHLH R genes in the precursor cells (Cave, 2005).

This study shows that there are important functional differences between the SPS architecture and non-SPS configurations of S binding sites. The SPS architecture is critical for synergistic activation of the m8 SPS+A bHLH R gene by Notch pathway and proneural proteins. Whereas previous studies have focused on which regulatory genes and proteins function combinatorially to activate SPS+A bHLH R gene expression, this study focuses on the underlying DNA transcription code that programs the Notch-proneural transcriptional synergy that drives cell-specific gene transcription. The results of previous studies have implied that an apparently arbitrary combination of S and A binding sites (S+A transcription code) is sufficient for transcriptional activation of SPS+A bHLH R genes. By contrast, this study shows that a minimal transcription code, SPS+A, is sufficient and critical for mediating Notch-proneural synergistic activation of these genes. The SPS+A code is composed of the specific SPS binding site architecture in combination with proneural A binding sites. Furthermore, evidence is presented that direct physical interactions between the Drosophila Su(H) and Daughterless protein mediate the transcriptional synergy, thus providing a molecular mechanism for the Notch-proneural synergy. Together, these studies show that the SPS architecture functions to mediate or enable the transcriptional synergy between Notch pathway and proneural proteins and that SPS+A is an architectural transcription code sufficient for cell-specific target gene activation during Notch signaling (Cave, 2005).

To test whether the SPS binding site architecture is important for Notch-proneural synergy, the ability of Drosophila NICD (dNICD) and proneural bHLH A proteins, such as Achaete and Daughterless (Ac/Da) to synergistically activate the wild-type native m8 promoter and SPS architecture variants was examined. Whereas the native m8 promoter carries the wild-type SPS architecture of S binding sites, the m8 promoter variants contain either a disrupted S site, leaving a single functional S site (S_F-X or X-S_R), or orientation variants in which the orientation of one or both S sites have been reversed (S_R-S_F, S_F- S_F, and S_R-S_R) (Cave, 2005).

The native m8 promoter is synergistically activated in transcription assays by coexpression of dNICD and Ac/Da, but it is only weakly activated by expression of dNICD or proneural Ac/Da proteins alone. However, neither promoter with a single S binding site (S_F-X or X-S_R) can mediate synergistic interactions between dNICD and proneural proteins. In fact, both single S site promoters are only weakly activated when proneural and dNICD proteins are expressed individually or together. Thus, single S sites are not sufficient to mediate Notch-proneural synergy in these contexts, even though they are in the same position as the SPS in the wild-type m8 promoter (Cave, 2005).

When the number of S binding sites are maintained, but the orientation of these sites within the SPS is varied (S_R-S_F, S_F-S_F, and S_R-S_R), only the wild-type (S_F-S_R) SPS orientation is synergistically activated by coexpression of dNICD and proneural Ac/Da proteins. Thus, the wild-type SPS architecture of S binding sites is clearly necessary for the m8 promoter to mediate transcriptional synergy between NICD and the proneural protein complexes assembled on the SPS and A sites, respectively (Cave, 2005).

The transcriptional synergy between NICD and proneural proteins mediated by the SPS element is crucial for the coactivation by the Mastermind (Mam) protein. Coexpression of Mam with both dNICD and proneural proteins provides a strong coactivation of transcription of the wild-type m8 promoter. However, this strong coactivation is not observed with any of the non-wild-type m8 SPS variants, which also cannot mediate Notch-proneural synergy. Thus, coactivation by both the NICD and Mam cofactors is strongly dependent on synergistic interactions with proneural combinatorial cofactors, and the specific SPS architecture is critical for mediating this synergy (Cave, 2005).

The native m8 promoter studies tested whether the organization of the S binding sites in the SPS are necessary to mediate the Notch-proneural synergy. In order to test which of these architectural features are sufficient to mediate that synergy, a set of synthetic promoters was created carrying the same SPS variants mentioned above in combination with A sites (SPS-4A reporter). These synthetic promoters thus contain the sites predicted to mediate the synergy but lack the other sites present in the native m8 promoter, which might also be necessary. This reductionist approach allows for the identification of a minimal promoter that contains only those sites that are necessary and sufficient to mediate the Notch-proneural synergy. All of these synthetic reporters are modestly activated by expression of proneural proteins alone, but expression of dNICD alone gives no activation. By contrast, only the SPS-4A reporter containing the wild-type SPS (S_F-S_R) mediates clear synergistic activation when dNICD and proneural proteins are coexpressed, and none of the SPS variants do so (Cave, 2005).

Given that functional CSL/NICD/Mam ternary complexes have been shown to assemble on single S sites and activate transcription, it was expected that promoters with single S sites could be activated at low levels by expression of dNICD in the absence of the proneural proteins and that promoters with two S sites might have more activity than single S sites. However, it was surprising to observe that all of the m8 and synthetic promoters, even with the wild-type SPS element, have very low or no activity when dNICD is expressed alone. Thus, the SPS binding site architecture does not appear to facilitate recruitment of functional NICD coactivator. This argues against previous proposals that suggested that the SPS architecture might function to recruit other proteins to the promoter. Thus, given that the wild-type SPS architecture is necessary and sufficient for Notch-proneural synergy, these results indicate that the function of the SPS element is to enable synergistic interactions with proneural proteins (Cave, 2005).

The synthetic promoters do not carry bHLH R sites, which are present in all E(spl)-C gene promoters. Thus, these sites clearly are not necessary for Notch-proneural synergy, although they may modulate it in vivo. It has been proposed that other repressor proteins bind the mγ and mδ SPS+A bHLH R gene promoters to restrict their expression to a subset of proneural clusters. Although these hypothetical repressor binding sites may be necessary to program the full mγ and mδ gene expression pattern, the current results indicate that they are not necessary for the Notch-proneural synergy that drives nonprecursor cell-specific upregulation (Cave, 2005).

Both the m8 and SPS-4A synthetic reporter contain a hexamer sequence that has been coconserved with the SPS element. Elimination of that hexamer site in a synthetic promoter does not disrupt Notch-proneural, suggesting that Notch-proneural synergy in vivo is not dependent on the hexamer site (Cave, 2005).

Together, the synthetic and m8 promoter results indicate that SPS+A is a minimal transcription code that is both necessary and sufficient for Notch-proneural synergy in Drosophila. The results with the promoters that were tested show that Notch-proneural transcriptional synergy requires the specific organization or architecture of the SPS element, in addition to its combination with proneural A binding sites. All of the promoters with SPS variants failed to mediate this synergy. This clearly indicates that arbitrary combinations of S and A binding sites are not sufficient to mediate Notch-proneural synergy (Cave, 2005).

An important question is whether there are other DNA binding transcription factors that can combinatorially synergize with CSL/NICD transcription complexes. Previous studies have shown that Notch pathway factors can synergize with a nonproneural transcription factor, Grainyhead, suggesting that synergy with the CSL/NICD transcription complexes could be very general or nonspecific. To test whether a general coactivator, the VP16 transcription activation domain, can synergistically interact with dNICD, an essentially identical wild-type SPS-containing synthetic promoter was created in which the A sites were replaced by UAS binding sites for the yeast Gal4 transcription factor (SPS-5U). Expression of a fusion protein containing the Gal4 DNA binding domain and the constitutively active VP16 activation domain can activate the synthetic SPS-5U promoter. However, the Gal4-VP16 fusion protein does not synergize with NICD. Thus, CSL/NICD complexes do not synergize with every nearby DNA bound transcription factor, and there is at least some specificity to the synergy with bHLH A proteins. This interaction specificity could contribute significantly to selective activation of Notch target genes. Further studies will be required to determine whether other DNA binding transcription factors can combinatorially synergize with Notch signaling and whether such factors fall into distinct classes (Cave, 2005).

Given that Notch signaling and neural bHLH A proteins have been conserved between Drosophila and mammals, it was next asked whether the transcriptional synergy between these proteins is also conserved in mammalian cells. Using the same set of synthetic promoters as mentioned above, activation following expression of the mammalian NICD and neural bHLH A protein homologs (Notch-1 ICD [mNICD] and MASH1/E47, respectively) was tested in murine NIH 3T3 cells. As in the Drosophila system, expression of MASH1/E47 proteins alone produces modest activation of the wild-type (S_F-S_R) SPS-4A promoter, and mNICD alone does not produce any significant activation of the promoter. However, clear transcriptional synergy is observed with the wild-type SPS promoter when both mNICD and neural bHLH A proteins are coexpressed. Moreover, SPS-mediated synergy requires nearly the same organizational features of S binding sites as observed in Drosophila. Neither of the single S site promoters can mediate that synergy, nor can most of the orientation variants. Although the S_R-S_R promoter is activated following coexpression of both the mNICD and bHLH A proteins, it is not activated by mNICD alone (Cave, 2005).

These results indicate that the potential for transcriptional synergy between NICD and neural bHLH A proteins has been conserved in a mammalian cell system and that the SPS+A code is sufficient and critical for mediating that transcriptional synergy. This raises the possibility that there may be mammalian genes that are regulated by neural bHLH A proteins and Notch signaling via this code. Although there is an SPS element conserved in the HES-1 promoter, HES-1 does not have an A site in its proximal promoter region, and HES-1 is not activated by expression of bHLH A genes. Thus, HES-1 appears to be similar to the Drosophila E(spl)-C m3 bHLH R gene, which also has an SPS but no obvious nearby A site. Whole-genome searches are being performed for genes in mammalian systems that may be regulated by the SPS+A code (Cave, 2005).

It has been proposed that the architecture of the SPS element may mediate cooperative binding of a second CSL protein once an initial CSL protein binds the DNA. Using electromobility gel shift assays to test for cooperative binding, the ability was compared of bacterially expressed and partially purified Drosophila Su(H) protein to bind DNA probes containing either the wild-type m8 SPS or an m8 SPS with one S site mutated. If there is cooperativity, one would expect to observe the band corresponding to two DNA bound CSL proteins to be as strong or stronger than the band corresponding to a single CSL protein bound to DNA. The single S site probe serves as a control because it cannot be cooperatively bound by two Su(H) proteins, and it also serves to identify the band corresponding to a single Su(H) protein bound to the wild-type SPS probe. Similar amounts of Su(H) protein bind strongly to the wild-type probe and to the single-site probe. In particular, because single protein binding to the wild-type DNA probe did not facilitate or stabilize simultaneous binding of two S proteins, Su(H) does not appear to bind cooperatively to the two S sites in the wild-type probe. These results suggest that CSL proteins do not bind cooperatively to the SPS in vivo, although posttranslational modifications in vivo could affect these binding properties Cave, 2005).

In addition, the protein binding affinity for the S_F-S_R and S_R-S_F probes appears to be comparable, although the reversed orientation of the two S sites would have likely disrupted cooperative binding if it were present. This result strongly suggests that the complete lack of activation by S_R-S_F sites in all of the promoters tested is not due simply to decreased ability of Su(H) protein to bind to the S_R-S_F orientation variant Cave, 2005).

To test the in vivo relevance of the conserved S binding site orientation in SPS elements, transgenic flies were created carrying β-galactosidase reporter genes driven by native m8 promoters containing either the wild-type (S_F-S_R) or S_R-S_F variant SPS elements. Wing and eye imaginal discs containing m8 promoters with the wild-type SPS element produced strong expression in proneural cluster regions, similar to the pattern described for endogenous m8. By contrast, comparably stained wing and eye discs carrying the m8 promoter reporters with the S_R-S_F SPS variant showed no expression or very low levels of expression, respectively. Extended staining of discs containing the S_R-S_F element revealed clear but weak expression in a pattern of single cells that resembles the distribution of neural precursors in the wing discs and eye discs. This is likely due to activation via the A site by proneural proteins because proneural levels are highest in the precursor cells. However, there was no expression in the surrounding nonprecursor cells within the proneural clusters even though Notch signaling is activated in these cells. Similar neural precursor-specific m8 reporter expression patterns have been observed when the S binding sites are eliminated, indicating that reversal of the S binding site orientations is functionally equivalent to eliminating them for this aspect of Notch target gene expression. These in vivo results confirm that the conserved orientation of the S binding sites in the wild-type SPS element is essential for nonprecursor cell specific upregulation of the SPS+A bHLH R m8 genes in response to Notch signaling in proneural clusters (Cave, 2005).

To gain an insight into the molecular mechanism underlying the strong transcriptional synergy between Notch signaling and bHLH A proteins on the m8 and SPS-4A promoters, whether this synergy involves a direct physical interaction was tested by using yeast two-hybrid assays with the Drosophila proteins. These experiments revealed that the Daughterless N-terminal domain directly and specifically interacts with the Su(H) protein in the absence of the bHLH domain and C terminus (Cave, 2005).

Using transcription assays in Drosophila cells, whether the Da N terminus (DaN construct), which contains a transcription activation domain, can synergistically activate the m8 promoter was tested in the absence of both its bHLH DNA binding domain and a heterodimerization partner, like Ac. The Da N-terminal protein synergistically activates the m8 promoter when dNICD is coexpressed, apparently by direct binding of the DaN protein to endogenous CSL bound to the SPS element. These results indicate that the Notch-proneural transcriptional synergy is not mediated by cooperative DNA binding interactions between the Su(H) and proneural proteins, although such cooperative binding may mediate transcriptional synergy between some combinatorial cofactors. These results suggest that a direct interaction between Su(H) and the Da N-terminal fragment, which can occur independent of NICD, facilitates the formation of an active transcription complex when NICD is also present during Notch signaling (Cave, 2005).

These results suggest that the SPS architecture functions to enable a direct physical interaction between Su(H) and Da proteins, thus providing a molecular mechanism for the observed Notch-proneural synergy that is mediated by the SPS element. This interaction could stabilize the recruitment or functional activity of NICD, which then recruits Mam, and could explain the strong dependence of both NICD and Mam coactivation functions on the presence of proneural proteins (Cave, 2005).

In previous studies, it has been proposed that neither the synergistic activation nor the transcriptional repression mediated by CSL protein complexes imply direct interactions between CSL and DNA bound combinatorial cofactors; rather, it is likely that CSL proteins exert their effects through the recruitment of non-DNA binding cofactors, such as chromatin modifying enzymes. While this might be the case for some Notch target gene promoters, in the case of m8, the results indicate that the mechanism underlying the synergistic interactions between CSL/NICD and bHLH A proteins does involve direct physical interactions (Cave, 2005).

A mechanistic model is proposed for programming Notch-proneural synergy with the SPS+A transcription code. These studies demonstrate that there are important functional differences between SPS and non-SPS organizations of S binding sites. The critical role of the SPS binding site architecture is not predicted or explained by the previous models for Notch target gene transcription. Previous models suggest that transcription is promoted by the binding of NICD to CSL, which displaces CSL bound corepressors, thus allowing transcriptional synergy with other DNA bound combinatorial cofactors. These models have not distinguished between Notch target genes with regulatory modules that contain SPS or non-SPS configurations of S binding sites, nor do they explain or predict the critical function of the SPS binding site architecture in mediating Notch-proneural transcriptional synergy (Cave, 2005).

A revised model is proposed that incorporates the essential requirement for the specific SPS binding site architecture in combination with the proneural A binding sites for transcriptional activation of m8 and the other SPS+A bHLH R genes. These genes each contain an SPS+A module and exhibit similar cell-specific upregulation in nonprecursor cells in proneural clusters. In this new model, the specific architecture of the S sites in the SPS element directs the oriented binding of Su(H) so that it is in the proper orientation and/or conformation to enable a direct interaction with Da. This interaction is an essential prerequisite for subsequent recruitment and/or functional coactivation by NICD during Notch signaling. This Notch-proneural complex is then further activated by subsequent recruitment of Mam (Cave, 2005).

It is interesting to note that the mammalian homologs of each of the Su(H), NICD, and Da proteins have been shown to interact with the p300 coactivator; thus, when complexed together, these proteins could potentially function combinatorially to recruit p300 or a related coactivator (Cave, 2005).

In Drosophila and mammals, Notch signaling is used throughout development to activate many different target genes, and in multiple developmental pathways. Thus, it is of paramount importance that the proper target genes are selectively activated in the proper cell-specific patterns. It is known that Notch signaling can activate genes through non-SPS configurations of S sites in certain other target genes. For example, expression of the Drosophila genes single minded, Su(H), and vestigal have all been shown to be regulated by Notch signaling, and all have single S sites or multiple unpaired S sites but no SPS elements in their promoter and/or enhancer regions (Cave, 2005).

The results show that for essentially every promoter tested, NICD cannot activate in the absence of neural bHLH A combinatorial cofactors, suggesting that NICD may always require a combinatorial cofactor to activate target genes. If so, the non-SPS Notch target genes are likely also to have specific combinatorial cofactors. The results also clearly show that the Notch-proneural combinatorial synergy requires a specific configuration of S sites, the SPS. There may be other specific configurations of S binding sites that mediate synergy for different classes of combinatorial cofactors for Notch signaling (Cave, 2005).

Together, these observations suggest that specific, but unknown, non-SPS configurations of sites may program the interactions between Notch complexes and the proper combinatorial cofactors. It is speculated that these non-SPS configurations might be unique to each target gene, or it is possible that there are specific patterns or classes of S binding site configurations -- an 'S binding site subcode' -- that determine cofactor specificity. Thus, the results suggest that selective Notch target gene activation may be programmed by distinct Notch transcription codes in which specific configurations of S binding sites mediate selective interactions with specific combinatorial cofactors (Cave, 2005).

Elucidating the various transcription codes controlling target gene activation during Notch signaling will be an important goal for future studies. The results have clearly shown that the architecture of transcription factor binding sites can be crucial for control of cell-specific Notch target gene activation. The studies presented here give a glimpse into the molecular mechanisms by which a one dimensional pattern of DNA binding sites can program cell-specific patterns of gene expression (Cave, 2005).

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).

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).

Drosophila LIM-only is a positive regulator of transcription during thoracic bristle development

The Drosophila LIM-only (Lmo) protein DLMO functions as a negative regulator of transcription during development of the fly wing. This study reports a novel role of Dlmo as a positive regulator of transcription during the development of thoracic sensory bristles. New dlmo mutants, which lack some thoracic dorsocentral (DC) bristles, were isolated. This phenotype is typical of malfunction of a thoracic multiprotein transcription complex, composed of Chip, Pannier (Pnr), Achaete (Ac), and Daughterless (Da). Genetic interactions reveal that dlmo synergizes with pnr and ac to promote the development of thoracic DC bristles. Moreover, loss-of-function of dlmo reduces the expression of a reporter target gene of this complex in vivo. Using the GAL4-UAS system it was also shown that dlmo is spatially expressed where this complex is known to be active. Glutathione-S-transferase (GST)-pulldown assays showed that Dlmo can physically bind Chip and Pnr through either of the two LIM domains of Dlmo, suggesting that Dlmo might function as part of this transcription complex in vivo. It is proposed that Dlmo exerts its positive effect on DC bristle development by serving as a bridging molecule between components of the thoracic transcription complex (Zenvirt, 2008).

The results presented in this study uncover a novel role of Dlmo in regulation of the development of the thoracic DC bristles. Homozygous, or hemizygous, loss-of-function (dlmo^hdp) mutants lack the anterior pair of the DC bristles. Moreover, these dlmo mutants displayed genetic interactions with mutants in genes known to regulate DC bristle development, such as pnr and ac, to reduce the number of DC bristles. Consistently, overexpression alleles of dlmo (dlmo^Bx) also exhibited genetic interactions with these pnr and ac mutants, resulting in an increased number of bristles. In addition, the finding that overexpression of pnr under the regulation of dlmo-GAL4 affects DC bristle development suggests that dlmo is expressed in the region of the wing disc that gives rise to these bristles (Zenvirt, 2008).

These results suggest a role of Dlmo in positive regulation of transcription. The negative role of Dlmo in modulation of transcription during Drosophila wing development has been well documented. The findings indicate that in another context, namely in regulation of DC bristle development by the Chip, Pnr, Ac and Da (CPAD) complex, Dlmo has another role, as a positive regulator of transcription. Lowering the level of Dlmo (in dlmo^hdp mutants) results in a reduction in the expression of a reporter driven by regulatory sequences of a bona fide target gene of the CPAD transcription complex, suggesting that Dlmo is a positive regulator of CPAD-dependent transcription. While the mechanism by which Dlmo positively regulates transcription in the context of the CPAD complex remains to be elucidated, a first clue to this mechanism may lie in the finding that Dlmo can bind constituent proteins of this complex, including Pnr and Chip, in vitro. Should these interactions also take place in vivo, Dlmo may exert its positive role in transcriptional regulation as a component of the CPAD complex (Zenvirt, 2008).

Insights into the mechanism of positive transcriptional regulation by Dlmo can be gleaned from LMO2, one of the mammalian homologs of Dlmo. LMO2 was demonstrated to participate in a multiprotein transcription complex that contains Ldb1, a GATA factor (GATA-1 or GATA-2), and the bHLH transcription factors TAL1 and E2A, which are homologous, respectively, to the fly components of the CPAD complex, Chip, Pannier, Achaete, and Daughterless. Various lines of evidence indicate that in mammals LMO2 serves as a bridge between components of the complex, and silencing of LMO2 causes disruption of the complex and decreases in the activation of transcription of its target genes, just as does silencing of Ldb1 or Tal1. Similarly to LMO2, Dlmo might serve as a bridge between components of the CPAD complex. LIM domains are protein-interaction modules and could serve Dlmo to bind components of the CPAD complex. This suggestion is supported by the finding that each single LIM domain of Dlmo is capable of binding components of the CPAD complex in vitro, and it agrees with similar reports on other LIM-containing proteins. Notably, a single LIM domain from LMO2 and LMO4 is sufficient to interact with Ldb1 or the related protein CLIM-1a. However, both LIM domains are required for the highest-affinity interactions (Zenvirt, 2008).

This proposed mode of action of Dlmo, as a bridging molecule, which binds a different protein through each one of its LIM domains, predicts that a Dlmo molecule with one defective LIM domain and one intact LIM domain would bind only one protein at a time and not be able to bridge between molecules. Indeed, in the new dlmo mutants it was found that deletions that span the second zinc finger of the second LIM domain of Dlmo, namely dlmo^hdp48-1 and dlmo^hdp185-1, resulted in dlmo loss-of-function mutations. These mutants display partial loss of thoracic DC bristles along with reduced expression of a target gene of the thoracic transcription complex. Interestingly, the wing size of these mutants is normal, unlike the small wings of mutants with lesions in the 5'-UTR of Dlmo, such as dlmo^hdp58-1, dlmo^hdp67-2, and dlmo^hdpR590. This may suggest that the defective Dlmo protein, which has only a single intact LIM domain, is sufficient for its function in the context of the wing, where Dlmo acts as a negative regulator that binds only one protein (CHIP), but is not sufficient when Dlmo acts as a bridging molecule in the thoracic CPAD transcription complex. Finally, the finding that Dlmo can bind other Dlmo molecules to generate homodimers or multimers might provide Dlmo with a greater flexibility of bridging between distant components of the complex. This possibility remains to be examined (Zenvirt, 2008).

In conclusion, Dlmo appears to have a dual role in regulation of transcription, depending on the context. Such a phenomenon has been documented for other transcription cofactors, whose dual function in transcription regulation varies according to their binding partners, the specific tissue, or the developmental stage. Likewise, these results indicate Dlmo has such a dual role, being a negative regulator with respect to the Ap-Chip complex and a positive regulator in the context of the CPAD complex (Zenvirt, 2008).

The Daughterless N-terminus directly mediates synergistic interactions with Notch transcription complexes via the SPS+A DNA transcription code

Cell-specific expression of a subset of Enhancer of split (E(spl)-C) genes in proneural clusters is mediated by synergistic interactions between bHLH A (basic Helix-Loop-Helix Activator) and Notch-signalling transcription complex (NTC) proteins. For a some of these E(spl)-C genes, such as m8, these synergistic interactions are programmed by an "SPS+A" transcription code in the cis-regulatory regions. However, the molecular mechanisms underlying this synergistic interaction between NTCs and proneural bHLH A proteins are not fully understood. Using cell transcription assays, it was shown that the N-terminal region of the Daughterless (Da) bHLH A protein is critical for synergistic interactions with NTCs that activate the E(spl)-C m8 promoter. These assays also show that this interaction is dependent on the specific inverted repeat architecture of Suppressor of Hairless (Su(H)) binding sites in the SPS+A transcription code. Using protein-protein interaction assays, it was shown that two distinct regions within the Da N-terminus make a direct physical interaction with the NTC protein Su(H). Deletion of these interaction domains in Da creates a dominant negative protein that eliminates NTC-bHLH A transcriptional synergy on the m8 promoter. In addition, over-expression of this dominant negative Da protein disrupts Notch-mediated lateral inhibition during mechanosensory bristle neurogenesis in vivo. These findings indicate that direct physical interactions between Da-N and Su(H) are critical for the transcriptional synergy between NTC and bHLH A proteins on the m8 promoter. These results also indicate that the orientation of the Su(H) binding sites in the SPS+A transcription code are critical for programming the interaction between Da-N and Su(H) proteins. Together, these findings provide insight into the molecular mechanisms by which the NTC synergistically interacts with bHLH A proteins to mediate Notch target gene expression in proneural clusters (Cave, 2009).

Essential roles of Da transactivation domains in neurogenesis and in E(spl)-mediated repression

E proteins are a special class of basic helix-loop-helix (bHLH) proteins that heterodimerize with many bHLH activators to regulate developmental decisions, such as myogenesis and neurogenesis. Daughterless (Da) is the sole E protein in Drosophila and is ubiquitously expressed. This study has characterized two transcription activation domains (TADs) in Da, called activation domain 1 (AD1) and loop-helix (LH), and has evaluated their roles in promoting peripheral neurogenesis. In this context, Da heterodimerizes with proneural proteins, such as Scute (Sc), which is dynamically expressed and also contributes a TAD. Either one of the Da TADs in the Da/Sc complex is sufficient to promote neurogenesis, whereas the Sc TAD is incapable of doing so. Besides its transcriptional activation role, the Da AD1 domain serves as an interaction platform for E(spl) proteins, bHLH-Orange family repressors which antagonize Da/Sc function. The E(spl) Orange domain is needed for this interaction and strongly contributes to the antiproneural activity of E(spl) proteins. A mechanistic model on the interplay of these bHLH factors in the context of neural fate assignment is presented (Zarifi, 2012).

It was hitherto believed that the main role of Da is to heterodimerize with other group A bHLH factors to enable them to bind their targets. This work has characterized two additional important functions of Da. First, its two transcriptional activation domains are critically needed for peripheral neurogenesis, whereas the TAD of the partner (Sc) is inactive in this context. Second, Da AD1 serves to recruit E(spl) proteins, which inhibit Da/Sc activity. This ability to recruit E(spl) is shared by the Sc TAD and contributes toward moderating the activity of proneural proteins, to restrict the number of SOPs formed in any given proneural field of cells. As AD1 and Sc TAD interact with different domains of E(spl) proteins, the Orange domain and the N terminus/bHLH, respectively, Da and Sc recruit a different subset of the seven E(spl) proteins (Zarifi, 2012).

One difference noted among the three Da/Sc TADs concerns their relative strengths, which seems to be tissue context sensitive. It is therefore possible that these conserved TADs have evolved for specific functions in different tissues. In vertebrate systems, there is a precedent for this; for example, the LH domain seems to be selectively active in myogenesis. Even within the same lineage, B lymphogenesis, the AD1 and LH of E2A are redundantly needed for some processes and differentially needed for others. In Drosophila, either of the two Da TADs (but not that of Sc) was able to sustain peripheral neurogenesis in the wing disk, antenna, and retina, despite the fact that when tested in isolation or in a simple reporter, they displayed disparate strengths (Zarifi, 2012).

The Da TADs could play a special role in neurogenesis if they recruit crucial coactivators for the induction of SOP-specific genes, such as ase, sens, and phyl, as these coactivators are dispensable for the EE4 enhancer. One characterized Da/Sc coactivator is Senseless. It is not thought that the recruitment of Sens is what distinguishes the Da TADs from the Sc TAD, as both Da and Sc have been shown to recruit Sens in glutathione S-transferase pull downs in vitro, whereas the current result would predict that only Da should be able to do so. Another transcription factor that can interact with each of the N-terminal two-thirds of Da (the AD1 and LH regions) is Su(H) (Cave, 2009). Although this interaction is important for the activation of E(spl)m8, which is broadly expressed in proneural territories, SOP-restricted Da/Sc target genes do not rely on Su(H) binding for activation. For these reasons, it is considered unlikely that either of the Da-Sens or Da-Su(H) interactions is what makes AD1 and LH necessary for SOP formation (Zarifi, 2012).

An alternative scenario for the necessity of the Da versus the Sc TADs in neurogenesis invokes the role of the Da REP domain. The latter consists of aa 503 to 535 and can downregulate the TAD function in cis, i.e., when juxtaposed to the AD1 or LH domains of the mammalian E-protein E2A/E12 (Markus, 2002), but also in trans, namely, on the Da partner Twist. If the latter is generally true, then perhaps Da/Sc dimerization inactivates the Sc TAD on some enhancers, making it unable to promote SOP-specific target gene transcription. This is consistent with the current results showing a dominant negative function of DaΔTADs (aa 415 to 710, which retain the REP domain) on processes that need either Ac-Sc or Ato activity. Cave (2009) constructed a more severely truncated Da molecule (DabHLH, aa 546 to 710), which lacks the REP domain, and did not report any dominant negative effects using the same C253-Gal4 driver as was used for the current. It should be stressed that the inhibitory effect of the REP domain appears to be target enhancer specific, although the mechanism behind this is still unexplored (Zarifi, 2012).

What is the role of the Da-E(spl) interaction? E(spl) proteins constitute a seven-member protein family of bHLH-O repressors that have arisen from recent gene duplication events in drosophilids and show partially redundant activities, with the most notable being the suppression of neural commitment, where they antagonize proneural proteins. This study showed that Da AD1 is an interaction surface for E(spl) proteins. A similar function has been shown for the Sc TAD. Both in vitro and in vivo suggest that these interactions serve to recruit E(spl) proteins onto Da/Sc-occupied enhancers rather than to prevent Da and Sc from binding onto DNA, which had earlier been proposed to be a mechanism of action for some of the vertebrate Hes proteins, homologues of E(spl) (Zarifi, 2012).

Besides interacting with Da and Sc, E(spl) proteins form potent DNA binding dimers that recognize binding sites distinct from those bound by Da/Sc. What, then, is the role of proneural-E(spl) protein interactions? Early insight on this was gained upon comparing bristle suppression by E(spl)m7 versus E(spl)mδ upon Sc overexpression. E(spl)m7, which interacts with both Da and Sc, can potently suppress bristle formation even at high Sc levels, whereas mδ (which does not interact with Da/Sc) can do so only at low (endogenous) Sc levels. The present work presents evidence that mutating the Orange domain of E(spl)m7 can compromise its bristle suppression activity without blocking its DNA binding-dependent repression activity. These results strongly suggest that most E(spl) proteins have evolved the ability to contact all three, DNA, Da, and Sc, in order to improve their recruitment onto enhancers. This is probably due to the extreme instability of E(spl) proteins, which prevents them from accumulating to high levels. In order for low E(spl) levels to have any biological significance, they would need to bind to target enhancers with high affinity. This would best be achieved by cooperative contributions from both DNA and protein contacts and would ensure robust repression of target genes over a broad range of Da/Sc levels. The efficiency of neural suppression will probably be the net result of DNA binding, Da binding, and Sc binding, although other factors such as intramolecular interactions may also contribute. This multiparameter system makes it hard to predict from only one feature (e.g., Orange domain integrity) the potency of bristle suppression by any given E(spl) variant (Zarifi, 2012).

The demonstration that Da AD1 interacts with the Orange domain of E(spl) proteins adds a new function to that elusive domain, which defines the bHLH-O subfamily and to date has been implicated in dimerization and Sens binding. The recently proposed structure of the Orange domain as a dimeric hairpin of two α helices is consistent with both its role in dimerization and its role as a protein interaction surface. In fact, both Sens and Da interactions have been mapped to the two extremes of the Orange domain, which are predicted to be in close proximity due to the folding back of the hairpin. Given its dual role in the selectivity of the bHLH-O dimer partner as well as in the binding to non-bHLH-O factors (such as Sens or Da), it comes as no surprise that the Orange domain had originally been recognized to be a specificity determinant for the function of bHLH-O repressors on the early Sxl enhancer (Zarifi, 2012).

E(spl) proteins are one (in fact, seven) out of a few negative regulators of Da/Sc and perhaps more group A bHLH activators, with others being Id/Emc and Eto/Nvy. E(spl) recruitment can account for some of the instances where Da has been shown to act repressively; e.g., E(spl) and Da have been shown to act interdependently to repress ato at the eye morphogenetic furrow. It should be noted that E(spl) uses a different mechanism from either of the other two negative regulators. Emc prevents Da DNA binding, whereas E(spl) does not; in fact, it seems to cooperatively enhance it. Nvy is a cofactor that is recruited onto Da but cannot bind DNA on its own. Given the nonconservation of Da AD1 with vertebrate E proteins but at the same time the conserved antagonism between E proteins and bHLH-O/Hes proteins in many contexts, it would be interesting to determine whether E-protein/Hes interactions can be detected in vertebrate systems and which mechanisms they use (Zarifi, 2012).

The bHLH transcription factor Hand is required for proper wing heart formation in Drosophila

The Hand basic helix-loop-helix transcription factors play an important role in the specification and patterning of various tissues in vertebrates and invertebrates. This study has investigated the function of Hand in the development of the Drosophila wing hearts which consist of somatic muscle cells as well as a mesodermally derived epithelium. Hand was found to be essential in both tissues for proper organ formation. Loss of Hand leads to a reduced number of cells in the mature organ and loss of wing heart functionality. In wing heart muscles Hand is required for the correct positioning of attachment sites, the parallel alignment of muscle cells, and the proper orientation of myofibrils. At the protein level, α-Spectrin and Dystroglycan are misdistributed suggesting a defect in the costameric network. Hand is also required for proper differentiation of the wing heart epithelium. Additionally, the handC-GFP reporter line is not active in the mutant suggesting an autoregulatory role of Hand in wing hearts. Finally, in a candidate-based RNAi mediated knock-down approach Daughterless and Nautilus were identified as potential dimerization partners of Hand in wing hearts (Togel, 2013).

In hand null mutants, wing hearts are formed but exhibit severe morphological defects resulting in loss of wing heart function. Consequently, almost all individuals display opaque wings and are unable to fly. Moreover, over time many of the mutant flies accumulate hemolymph in their wings. This long term effect occurs also very frequently in flies that completely lack wing hearts. During wing inflation, hemolymph is forced into the wings by elevated hemolymph pressure in the thorax which is effectuated by rhythmic contractions of the abdomen. However, this does not result in uncontrolled hemolymph accumulation as observed in animals lacking wing heart function since the epidermal cells of the wings still interconnect the opposing wing surfaces at this stage. Only after their delamination, more hemolymph may accumulate resulting in balloon-like wings which explains the long term character of this phenotype. However, since a rather large amount of hemolymph may accumulate in the wings some mechanism must exist that prevents backflow into the body cavity. In the tubular connection between wing and wing heart, a back-flow valve exists that prohibits hemolymph flow from the body cavity into the wing and thus is unsuitable to maintain a large amount of hemolymph inside the wing. In the region of the hinge, no valves are present and hemolymph may freely enter or leave the wings. It is therefore assumed that the apoptotic epidermal cells that remain in the wings due to loss of wing heart function form clots in the inflow and outflow tracts and thereby block hemolymph passage. Animals exhibiting these long term effects are probably affected in various ways. Most obviously, flies with filled wings have difficulties moving around and tend to fall during climbing. However, there are probably also physiological effects since the amount of hemolymph trapped in the wings must be lacking in the body cavity and should therefore affect internal hemolymph pressure as well as tissue homeostasis. Thus, it is proposed that the long term effects on wing morphology may contribute to the observed shortened life span of adult hand mutants (Togel, 2013).

Based on handC-GFP reporter activity, wing hearts express hand throughout their entire development and probably also during their mature state. However, the requirement for Hand seems only critical during early pupal stages at the time when the wing hearts are formed. Similarly, hand mutants display a phenotype in the adult only with regard to the heart and the midgut indicating that Hand is likewise required only during metamorphosis in these organs. In the adult heart, loss of hand leads to disorganized myofibrils, a phenotype that was also observed in mature wing heart muscles. Additionally, it was found that the attachment sites are less regular leading to a disruption of the dorso-ventral order of the muscle cells and loss of their parallel alignment. In many cases, muscle cells even form ectopic attachment sites in an area where they never occur in the wild-type. In an attempt to characterize the phenotype at the protein level, it was found that α-Spectrin and Dystroglycan are not properly distributed. Both proteins constitute components of the costameric network and are enriched at the membrane overlying Z-discs in the wild-type. In hand mutants, however, their pattern is altered to a more or less homogenous distribution at the membrane. In knock-downs of α- or β-Spectrin in the postsynaptic neuromuscular junction (NMJ), it was shown that spectrins are required for normal growth of NMJs and normal distribution of Dlg at the junctions. The enlarged NMJs visible in the Dlg staining, support the observation that α-Spectrin is misdistributed in hand mutants. The similar localization of α-Spectrin and Dystroglycan at the membrane raises the question whether their misdistribution in the mutant is somehow interconnected or independent of each other. Spectrins are organized in tetramers, consisting of two α/β-Spectrin heterodimers, which bind actin and are connected to the plasma membrane via Ankyrins. Ankyrins, in turn, have binding sites for Dystroglycan and E-Cadherin and together Ankyrin and the actin/spectrin network are thought to stabilize cell-cell and cell-matrix attachments. A hint that the misdistribution of α-Spectrin and Dystroglycan may be interconnected comes from observations in Dystrophin deficient mdx mice. There, Dystroglycan and β-Spectrin are both irregularly distributed but always co-localize. This let the authors conclude that their organization is coordinated. A possible explanation for the general loss of costamere organization may be that the costameric γ-Actin, although expressed normally, does not form a stable link between Z-discs and the membrane in mxd mice. However, loss of Dystrophin results mostly in the disruption of the linear arrangement of the proteins at the Z- and M-lines and does not lead to their homogenous distribution as observed in hand mutants. Nevertheless, the data obtained in this study and the phenotypic analysis of loss of function studies strongly suggest that the Spectrin and Dystroglycan phenotypes in hand mutants are interconnected caused by a, yet unknown, defect in the costameric network. Moreover, the misdistribution of these two proteins suggests that other proteins might be affected in a similar way including receptors required for directed outgrowth of muscle cells and proper targeting of tendon cells. This would explain why many muscle cells attach at improper positions or are misaligned. However, the cytoskeleton is not affected as a whole since the muscle cells still attain an elongated shape with attachment sites at their ends and a wild-typic βPS-Integrin pattern (Togel, 2013).

In the epithelium, loss of hand results in the failure of cells to integrate into the developing epithelium leading to gaps and the loss of cells. In mature organs, cells are predominantly missing in the area that dorsally extends the muscle cells suggesting that epithelial cells have greater difficulties attaching to their own type than to the muscle cells. On the protein level, it was found that Arm does not localize to the periphery of the cells except for small dot-like areas in the remaining filopodia-like cellular interconnections. Arm (β-Catenin) constitutes an intracellular adapter protein that links the transmembrane receptor E-Cadherin to actin filaments in adherens junctions. Adherens junctions are predominantly found between cells of the same type whereas Integrin based hemiadherens junctions connect to the ECM and additionally form specialized junctions between different cell types (e.g. myotendinous junctions). Based on the correct distribution of βPS-Integrin and the absence of Arm at the cell borders in hand mutants, it could be that the epithelial cells are able to form hemiadherens junctions towards the muscle cells but fail to establish a sufficient number of adherens junctions towards other epithelial cells. However, an alternative explanation would be that the formation of hemiadherens junctions is not affected and the remaining cells are simply too far apart to establish proper cell-cell contacts. Further experiments are needed to clarify this point (Togel, 2013).

It has been suggested that Hand proteins are involved in the inhibition of apoptosis based on the observation that loss of Hand function leads to hypoplasia and that block of apoptosis in the mutant background, at least partially, rescues the hand phenotype. This study observed a similar effect with respect to wing heart cell number. However, live cell imaging showed that also in the wild-type muscle cells are removed by apoptosis suggesting that this is a normal process during regulation of muscle cell number. Consequently, block of apoptosis in the controls led to an increase in muscle cell number. This suggests further that wing hearts in general have the potential to form more functional muscle cells. In hand mutants, the same removal of muscle cells occurs indicating that hand does not in general block apoptosis in wing hearts. Moreover, since the inhibition of apoptosis by P35 also affects the apoptosis involved in regulation of muscle cell number it cannot be excluded that the observed effect is actually induced hyperplasia in the mutant background mimicking a rescue instead of a real rescue of the hand phenotype. Additionally, live cell imaging showed that the cells of the wing heart epithelium forming the dorsal extension arise at their correct position in a sufficient number so that no gaps are visible. Only after they fail to establish proper cell–cell contacts they are removed from the wing hearts. It is therefore proposed that loss of cells by apoptosis in hand mutants is only a secondary effect caused by the inability of cells to integrate into the forming wing hearts (Togel, 2013).

It has been shown that Hand proteins can function as transcriptional activators in vertebrates and invertebrates. However, no direct targets have been identified in Drosophila so far. This study reports that the handC-GFP reporter line shows almost no activity in wing heart progenitors during postembryonic development suggesting that hand itself is a direct target of Hand. Remarkably, in some individuals a few nuclei of the wing hearts still show reporter activity indicating that hand is not the only transcription factor involved in postembryonic activation of the reporter. Moreover, the fact that the hand null mutant is not always a null with respect to reporter activation makes it a variable phenotype. Similarly, the severity of the phenotype observed in individual wing hearts (e.g. left and right side of the same animal) may differ considerably. So, how can a null mutation cause variable phenotypes? The answer may lie in the fact that all bHLH transcription factors need to form homo- or heterodimers for DNA binding. It was therefore proposed that the absence of a bHLH transcription factor not only affects its direct downstream targets but also the entire bHLH factor stoichiometry within the cell suggesting that the pool of bHLH dimers might be dynamically balanced. In the absence of Hand, new and presumably also artificial bHLH dimers are formed which consequently can cause a variety of delicate differentiation defects. The scenario is becoming even more complicated by the observation that the dimerization property of Hand is modulated by its phosphorylation state as well as by the finding that Hand can inhibit the dimerization of other transcription factors by blocking their protein interaction sites (Togel, 2013).

A crucial prerequisite for understanding the bHLH network in wing hearts is therefore the identification of dimerization partners. In a candidate based RNAi approach, two bHLH proteins, Da and Nau, were identified which evoke a phenotype very similar to the hand mutant. In order to verify the indication that these factors are interacting with Hand, Y2H analysis was applied and and an interaction between both Hand and Da as well as Hand and Nau was confirmed at the protein level. Furthermore, in vertebrates it was shown that these proteins are also able to form heterodimers with each other and that Hand is able to compete for heterodimer formation and DNA binding. Thus, based on Y2H interaction as well as phenotype similarity, the potential bHLH network in wing hearts likely includes Hand/Da and Hand/Nau heterodimers which activate different sets of downstream genes. In hand null mutants, the balance may be shifted to Da/Nau heterodimers or even Da/Da or Nau/Nau homodimers which may be able to activate some of Hand's target genes but with lower or higher efficiency. The competition of all these dimers with different transcriptional activation efficiency for the hand targets might explain the variations observed in the hand mutants (Togel, 2013).

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