InteractiveFly: GeneBrief

Enhancer of split m4, Bearded family member : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Enhancer of split m4, Bearded family member

Synonyms - E(spl) region transcript m4, m4

Cytological map position - 96F9

Function - signaling protein

Keywords - opposes action of Notch pathway

Symbol - E(spl)m4-BFM

FlyBase ID: FBgn0002629

Genetic map position - 3-89.1

Classification - basic amphipathic alpha-helix related to Bearded

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

The E(spl) locus, located at 96F8-14, harbors 12 transcription units: malpha, mbeta, mdelta, mgamma, and m1, m2, m3, m4, m5, m6, m7 and m8. Molecular analysis has shown that seven of these genes, namely mbeta, mdelta, mgamma, m3, m5, m7 and m8, encode highly related basic-helix-loop-helix (bHLH) proteins, and are turned on in response to Notch signaling, via the transcriptional mediator Su(H). Since E(spl) bHLH proteins are able to act as transcriptional repressors of proneural genes, they probably act as endpoint effectors in the suppression of neural fate mediated by Notch. The pattern of expression of the E(spl)bHLH genes agrees with this model, since it closely matches the domains of neurogenesis, both in the embryo and the larva. The roles of the non-bHLH genes of the E(spl) region have not been fully characterized to date. However, at least one of them, m4, has been long known to have a similar expression pattern to that of E(spl) bHLH genes and appears to also be inducible by Notch signaling in a Suppressor of Hairless-dependent manner (Knust, 1987; Singson, 1994; Bailey, 1995). The whole locus has been scanned for genes showing transcriptional response to activated Notch. In addition to showing that all seven bHLH genes can be turned on by ectopic expression of a constitutively active form of Notch, an identical behaviour has been documented for some of the non-bHLH genes. Could these proteins act in conjunction with the E(spl) bHLH factors to oppose neurogenesis (Wurmbach, 1999).

Among the non-bHLH Notch-inducible genes of the E(spl) Complex are m4 and malpha. The embryonic expression pattern of both m4 and malpha is remarkably similar to that of E(spl)bHLH. In imaginal disks they both show strong expression in areas where peripheral neural elements arise, e.g. the macrochaete proneural clusters of the late larval wing disk, with malpha displaying additional expression at other sites (Wurmbach, 1999). Interestingly, Bearded (Brd) (Leviten, 1997), which maps outside the E(spl) locus, encodes a product with weak structural similarity to the N-terminal half of m4 (31% identical; 48% similar) and malpha (25% identical/37% similar). While it is not known whether Brd is transcriptionally activated by Notch signaling, it has been shown to participate in neural fate acquisition. The products encoded by these genes display significant sequence similarity to each other (44% identity, 66% similarity) (Wurmbach, 1999). Apparent paralogs of E(spl)m4 have been named Bob ( Brother of Bearded) and Tom (Twin of m4), respectively. The Bearded (Brd) family proteins can be classified as Brd-like (Brd and Bob) or m4-like (m4, malpha, and Tom), based on their relative sizes and degree of amino acid similarity (For information about Bob and Tom see the m4 Evolutionary Homologs section). Hypermorphic Brd alleles produce supernumerary external sensory bristles in the adult and display genetic interactions which argue for a model where Brd counteracts the activity of the Notch pathway in sensory organ specification. If m4 and malpha have a function similar to Brd, they should promote neural fate: this is contrary to expectation for a Notch-driven class of genes, which oppose neural fates. Unlike their neighboring E(spl)bHLH genes, overexpression of either m4 or malpha appears to counteract Notch signaling; the overexpression produces supernumerary chaetae, a phenotype similar to that of Brd gain-of-function (gof) alleles. These arise from suppression of the Notch-mediated lateral inhibition process, because their progenitor cells (the sensory organ precursors, or SOPs) are seen in close apposition to one another in imaginal disc epithelia. The possible mechanism for the function of these proteins has been analyzed, as well as their relationships to various components of the Notch signaling pathway (Apidianakis, 1999).

Intercellular signaling mediated by Notch proteins is crucial to many cell fate decisions in metazoans. Its profound effects on cell fate and proliferation require that a complex set of responses involving positive and negative signal transducers be orchestrated around each instance of signaling. In Drosophila the basic-helix-loop-helix (bHLH) repressor encoding genes of the E(spl) locus are induced by Notch signaling and mediate some of its effects, such as suppression of neural fate. A novel family of Notch responsive genes, whose products appear to act as antagonists of the Notch signal in the process of adult sensory organ precursor singularization, has been described. They, too, reside in the E(spl) locus and comprise transcription units E(spl) m4 and E(spl) malpha. Overexpression of these genes causes downregulation of E(spl) bHLH expression accompanied by cell autonomous overcommitment of sensory organ precursors and tufting of bristles. Interestingly, negative regulation of the Notch pathway by overexpression of E(spl) m4 and malpha is specific to the process of sensory organ precursor singularization and does not impinge on other instances of Notch signaling (Apidianakis, 1999).

Experiments by Nagel (2000), suggest that the overexpression phenotype of E(spl) m4 and E(spl) malpha obtained by Apidianakis (1999) is likely to be due to a dominant negative effect and does not reflect the biological function of these two genes. In order to elucidate m4/malpha gene function directly, RNAi, which causes sequence-specific transcript degradation, was carried out by injecting either m4 or malpha double-stranded RNA or a mixture of both into pre-blastoderm embryos. In agreement with genetic data, RNAi causes a high incidence of lethality (~50%). Dead embryos develop intermediate to strong neurogenic phenotypes (too many neurons) typical of loss of E(spl) bHLH activity. Surviving embryos hatch into wild type appearing larvae that develop normally to adult flies. From this it is concluded that the m4/malpha genes are required to positively transduce the Notch signal during neurogenesis, and presumably during bristle development as well. Therefore, suppression of lateral inhibition observed after overexpression of either m4/malpha family member must be due to a dominant-negative effect, presumably by titrating out other important Notch pathway components (Nagel, 2000).

A third member of the m4/malpha gene family, bbu/tom, has been identified and has been localized within the Brd-C (see Bearded). It is of interest to know whether bbu/tom has a function during Drosophila development similar to m4/malpha. Overexpression of bbu/tom causes bristle tufting phenotypically indistinguishable from m4/malpha. The molecular mechanism underlying bristle tufting is also based on interference with E(spl) gene activity in a similar manner as occurs for m4/malpha. Ectopic expression of either bbu/tom, m4 or malpha results in the selection of additional SOPs due to silencing of, for example, E(spl) m8 transcription. Repression is specific to those E(spl) genes that are involved in bristle specification and does not include mbeta, which acts mainly in wing vein development. Apparently, bbu/tom overexpression has identical effects on the development of mechanosensory bristles as that of m4/malpha. It was thus of interest to see whether bbu/tom is involved in neurogenesis in the same way, and this was again analyzed by dsRNA injections. Lethality was induced at a similar rate, but neurogenic embryos were much rarer. Instead, a large fraction of the dead embryos did not develop to a stage where they secrete cuticles. The most conspicuous phenotypes of the older embryos were defects during head involution. Thus, bbu/tom is expected to play a role different from m4/malpha during Drosophila embryonic development (Nagel, 2000).

The fact that the activity of bbu/tom differs clearly from either m4 or malpha might be surprising because all three genes are robustly expressed in the neuroectoderm in germ band extended embryos. It is noted, however, that this is the major region of co-expression of the three genes, which show distinct expression patterns during earlier and later stages of embryogenesis. Up to the blastoderm stage, bbu/tom transcripts accumulate in the presumptive ectoderm and are excluded from the presumptive mesoderm. In contrast, m4/malpha are exclusively expressed in the mesectoderm at that stage. Starting at germ band retraction, bbu/tom mRNA is enriched along the midline and in the segmental folds within the lateral ectoderm. The pattern is also quite distinct in wing and leg imaginal discs, whereas in the eye disc, expression bordering the morphogenetic furrow is reminiscent of m4/malpha. No induction of bbu/tom transcription is observed by ectopic expression of the activated Notch receptor in a pair rule pattern in the embryo (prd-Gal4 >>UAS-Nintra). Furthermore, transcriptional activation of bbu/tom is not dependent on Su(H) because Su(H) mutant embryos derived from germ line clones still express bbu/tom normally in the lateral ectoderm. The absence of midline staining is attributed to the hypertrophic nervous system in these embryos (Nagel, 2000).

What is the physiological role of the m4/malpha family? The m4/malpha family contains three members of high structural similarity and up to eight, taking the related Brd gene family into account. Overexpression of various members gives surprisingly similar phenotypes -- ectopic bristles (mechanosensory organs) that result from a failure of the lateral specification of singular bristle precursor cells (SOPs). From these gain of function phenotypes, it has been concluded that this class of gene plays a negative role in Notch-mediated signaling processes. A lack of phenotypes in loss of function mutations of single genes gives little genetic support to this hypothesis and favors functional redundancy between the individual genes (Nagel, 2000).

Despite the similarity between these genes, there is a major difference between the members of the m4/malpha and Brd gene families, in that only m4 and malpha are direct transcriptional targets of Notch signaling whereas genes in the Brd-C are not. De-repression of bbu/tom transcription within the ventral ectoderm after driving Notch with a Kr-Gal4 line appears to conflict with the lack of transcriptional activation by Notch that has been observed. One possibility to resolve this conflict is to note that Kr-Gal4 is expressed earlier than prd-Gal4. Since Notch is notorious for its ability to affect cell fate at many stages, it is not unlikely that the earlier ectopic expression might cause a cell fate change of the ventral ectoderm to lateral ectoderm, where bbu/tom is strongly expressed. Put more parsimoniously, the ectopic activation of bbu/tom expression is most likely a secondary indirect effect, since it is seen when Nintra is driven by one Gal4 line and not by another. Because the transcriptional regulation of these genes is different, it is quite likely that their physiological roles might also be different. The RNAi experiments now reveal a role for m4/malpha as positive factors in the Notch pathway during neurogenesis in the early embryo, whereas bbu/tom appears to function differently. This interpretation is corroborated by genetic evidence using a combination of small deletions in the E(spl)-C that result in failure of lateral inhibition during neurogenesis as well as bristle development. Whereas deficiencies in m4 increase bristle density, those in Brd and bbu/tom do not, even in the background of E(spl)-C deficiencies. Hence, despite the structural similarities between members of the m4/malpha family, bbu/tom has lost a number of their characteristics, most notably Notch responsiveness in agreement with its remote chromosomal position. The phenotypes achieved by overexpression of any of the three genes, however, are very similar. They are likely a result of dominant-negative effects and thus do not allow conclusions to be drawn on a role for this gene family in normal bristle development. They rather indicate an ability of this class of proteins to interact with other factors specifically required during the process of bristle precursor specification (Nagel, 2000).


REGULATION
Promoter Structure

The functional relationships have been investigated among three loci that are required for multiple alternative cell fate decisions during adult peripheral neurogenesis in Drosophila: Notch (N), which encodes a transmembrane receptor protein; Suppressor of Hairless [Su(H)], which encodes a DNA-binding transcription factor, and the Enhancer of split gene complex [E(spl)-C], which includes seven transcription units that encode basic helix-loop-helix (bHLH) repressor proteins. Several lines of evidence are described establishing that Su(H) directly activates transcription of E(spl)-C genes in response to N receptor activity. Expression of an activated form of the N receptor leads to elevated and ectopic E(spl)-C transcript accumulation and promoter activity in imaginal discs. The proximal upstream regions of three E(spl)-C genes, including m4, contain multiple specific binding sites for Su(H). The integrity of these sites, as well as Su(H) gene activity, are required not only for normal levels of expression of E(spl)-C genes in imaginal disc proneural clusters, but also for their transcriptional response to hyperactivity of the N receptor. Under Su(H) minus conditions, both the endogenous m4 gene and the 51-bp m4 promoter fragment exhibit strong expression in PNS proneural clusters; in the latter case at least, the expression is dependent on the integrity of two E-box binding sites for proneural activators. These results establish Su(H) as a direct regulatory link between N receptor activity and the expression of E(spl)-C genes, extending the known linear structure of the N cell-cell signaling pathway (Bailey, 1995 and Nellesen, 1999).

In Drosophila, genes of the Enhancer of split Complex [E(spl)-C] are important components of the Notch (N) cell-cell signaling pathway, which is utilized in imaginal discs to effect a series of cell fate decisions during adult peripheral nervous system development. Seven genes in the complex encode basic helix-loop-helix (bHLH) transcriptional repressors, while four others encode members of the Bearded family of small proteins. A striking diversity is observed in the imaginal disc expression patterns of the various E(spl)-C genes, suggestive of a diversity of function, but the mechanistic basis of this variety has not been elucidated. Strong evidence is presented from promoter-reporter transgene experiments that regulation at the transcriptional level is primarily responsible. Certain E(spl)-C genes are known to be direct targets of transcriptional activation both by the N-signal-dependent activator Suppressor of Hairless [Su(H)] and by the proneural bHLH proteins achaete and scute. Extensive sequence analysis of the promoter-proximal upstream regions of 12 transcription units in the E(spl)-C reveals that such dual transcriptional activation is likely to be the rule for at least 10 of the 12 genes. The very different wing imaginal disc expression patterns of E(spl)m4 and E(spl)mgamma are a property of small (200-300 bp), evolutionarily conserved transcriptional enhancer elements, which can confer these distinct patterns on a heterologous promoter despite their considerable structural similarity [each having three Su(H) and two proneural protein binding sites]. As originally defined by its structure in m4, m8 and mgamma, the Su(H) paired site (SPS) configuration consists of two high-affinity (YGTGRGAAM; M denotes A or C) Su(H) binding sites in an inerted repeat arrangement, with 300 bp between the first G of the two sites. In addition, the 'Y' of the upstream site is T, while that of the downstream site is C. Finally, the sequence between the two Su(H) sites includes the hexamer GAAAGT or its complement ACTTTC. The SPS motifs of m4 and mgamma in Drosophila hydei are remarkably conserved. A 43-nt block in m4 SPS contains the entire SPS motif and shows only a single varient position. Also all four bHLH activator binding sites in the two genes are conserved. A putative bHLH repressor binding site defined by the N box consensus sequence CACNAG does not appear to be as conserved as sites defined by CACGYG that have been shown to bind these proteins with high affinity. Thus of four distinct CACGYG sites (one in mgamma and three in m4 ), two are conserved, while apparently only one of seven distinct N box sites (three in mgamma and four in m4) is conserved. Conserved transcription factor binding sites are often accompanied by strongly conserved flanking sequences. It is concluded that the distinctive expression patterns of E(spl)-C genes in imaginal tissues depend to a significant degree on the capacity of their transcriptional cis-regulatory apparatus to respond selectively to direct proneural- and Su(H)-mediated activation, often in only a subset of the territories and cells in which these modes of regulation are operative (Nellesen, 1999).

Transcriptional Regulation

The neurogenic genes of Drosophila are required for correct separation of neural and epidermal progenitor cells during early embryogenesis. Results from genetic analyses indicate that the neurogenic genes are functionally related. The spatial distribution of RNA from the neurogenic genes Dl, neu, and m4, m5, m7 and E(spl) [four genes of the Enhancer of split complex] has been studied in various neurogenic mutant embryos by in situ hybridization. An abnormal distribution of RNA from certain of the genes is found in neurogenic mutants, suggesting that at least some of the functional interactions inferred from genetic data take place at the transcriptional level (Godt, 1991).

Transcriptional expression of some E(spl)-C genes encoding bHLH transcription factors is activated in response to Notch signaling. In order to elucidate whether there are additional Notch responsive genes among the newly characterized genes, all bHLH and non-bHLH genes in the E(spl) locus were systematically compared for their Notch responsiveness. Truncation of the extracellular domain of Notch results in a constitutively activated Notch receptor. Such an activated Notch receptor (N intra) was expressed in a zebra pattern employing the Gal4/UAS system. These embryos were subsequently hybridized with probes specific for the particular genes of the E(spl) locus. If the transcription of the respective gene is dependent on the Notch signal, it should be expressed in a striped pattern. All genes within the E(spl) locus except m1 and m6 are activated in seven stripes, indicating that these genes are regulated by the Notch pathway during embryogenesis. From this it is concluded that the E(spl)-C comprises three different classes of Notch responsive genes: the seven bHLH genes; malpha/m4, and m2 (Wurmbach, 1999).

The transcription factor Su(H) seems to provide a direct link between the activated Notch receptor and Notch target genes. In accordance, the promoter regions of the E(spl)-C genes mdelta, mgamma, m4, m5 and m8 contain respective Su(H) protein binding sites. An investigation was carried out to see whether this is also true for malpha and m2. Embryos homozygous mutant for Su(H) were derived from germ-line clones. These Su(H) mutant embryos were hybridized in situ with probes for m2, malpha, and m4 (as control), respectively, and compared with the heterozygous siblings that bear one Su(H) zygotic copy and, thus, develop normally. malpha and m4 transcripts are nearly absent in the neuro-ectoderm of the mutant embryos. This result is very similar to that observed for E(spl) bHLH genes. In accordance, the malpha promoter region contains two putative Su(H) binding sites that fit exactly the consensus sequence [(C/T)GTG(G/A)GAA(C/ A)], and two potential sites that differ in their last position (T instead of C/A at position 9), a variation observed before for a genuine Su(H) binding site in the md promoter. Similarly, the promoter region of m4 contains three perfect Su(H) binding sites (Bailey, 1995 and Wurmbach, 1999).

In contrast to m4 and malpha, a considerable amount of m2 transcript is found in Su(H) mutant embryos especially in the dorsal ectoderm, although at a reduced level. Apparently, Su(H) is not strictly required for the initiation of m2 transcription, but seems to augment it. Only at later stages of development, m2 transcripts disappear, suggesting that Su(H) is essential for the maintenance of m2 transcription. The m2 promoter region contains a single divergent Su(H) binding site that differs at two positions, the fifth and the ninth, from the consensus. Similarly divergent Su(H) binding sites have been shown to bind the Su(H) protein in vitro (Wurmbach, 1999).

Targets of Activity

The transcriptional effect of m4/alpha is observed in S2 cultured cells, where two different luciferase reporter genes, one driven by a promoter fragment of E(spl)- mgamma and one by a promoter fragment of E(spl)-m8 were tested. These promoters are induced to 6x and 12x, respectively, after cotransfection with plasmids expressing Su(H) and an intra-cellular activated form of Notch (RICN). Addition of expression plasmid for m4 or malpha gives a small yet consistent ~2x repression of both reporters. In contrast, m4/alpha has no effect on an unrelated luciferase reporter gene driven by the achaete proximal promoter. A specific repressive effect of m4/alpha is thus observed on E(spl)bHLH transcription both in imaginal discs and in cultured cells. The ability of m4/alpha to downregulate E(spl)bHLH levels, could account for their promotion of SOP fate. If this is the sole activity of m4/alpha proteins, then restoring the expression of E(spl)bHLH genes should abolish the m4/alpha gain-of-function (GOF) phenotype. Scutellum specific GAL4 lines were used to ectopically co-express m4 or malpha with one of the E(spl)bHLH genes. Whereas m4/alpha alone produces 6+/-13 scutellar bristles, coexpression of E(spl)bHLH eliminates scutellars, a phenotype indistinguishable from the gain-of-function phenotype of E(spl)bHLH alone. This epistatic relation of UAS-E(spl) over UAS-m4/alpha led to the conclusion that m4/alpha cannot antagonize E(spl) bHLH factors at the protein level; rather, they must act upstream of E(spl) bHLH accumulation. Consistent with this conclusion, no interactions is observed for m4 or malpha with a number of E(spl) bHLH or with their co-repressor Groucho in a yeast two-hybrid assay. Furthermore, m4/alpha does not interact with the proneural proteins Da, Ac, Sc or Ato. In fact m4 appears to be a cytoplasmic protein, at least as far as can be judged from the subcellular localization of an m4- green fluorescent protein (GFP) fusion, making interaction with these nuclear factors unlikely (Apidianakis, 1999).

Post-transcriptional Regulation

During the development of the Drosophila adult peripheral nervous system (PNS), inhibitory cell-cell interactions mediated by the Notch receptor are essential for proper specification of sensory organ cell fates. The 3' untranslated regions (UTRs) of many genes involved in Notch signaling, including Bearded (Brd) and the genes of the Enhancer of split Complex (E(spl)-C), contain (often in multiple copies) two novel heptanucleotide sequence motifs: the Brd box (AGCTTTA) and the GY box (GTCTTCC). Moreover, the molecular lesion associated with a strong gain-of-function mutant of Brd suggests that the loss of these sequence elements from its 3' UTR might be responsible for the hyperactivity of the mutant gene. The wild-type Brd 3' UTR confers negative regulatory activity on heterologous transcripts in vivo and this activity requires its three Brd box elements and, to a lesser extent, its GY box. Brd box-mediated regulation decreases both transcript and protein levels, and the results suggest that deadenylation or inhibition of polyadenylation is a component of this regulation. Though Brd and the E(spl)-C genes are expressed in spatially restricted patterns in both embryos and imaginal discs, the regulatory activity that functions through the Brd box is both temporally and spatially general. A Brd genomic DNA transgene with specific mutations in its Brd and GY boxes exhibits hypermorphic activity that results in characteristic defects in PNS development, demonstrating that Brd is normally regulated by these motifs. Brd boxes and GY boxes in the E(spl)m4 gene are specifically conserved between two distantly related Drosophila species, strongly suggesting that E(spl)-C genes are regulated by these elements as well (Lai, 1998a).

Most E(spl)-C genes contain a novel sequence motif, the K box (TGTGAT), in their 3' untranslated regions (3' UTRs). Three lines of evidence are presented that demonstrate the importance of this element in the post-transcriptional regulation of E(spl)-C genes. (1) K box sequences are specifically conserved in the orthologs of two structurally distinct E(spl)-C genes (m4 and m8) from a distantly related Drosophila species; (2) the wild-type m8 3' UTR strongly reduces accumulation of heterologous transcripts in vivo, an activity that requires its K box sequences, and (3) m8 genomic DNA transgenes lacking these motifs cause mild gain-of-function PNS defects and can partially phenocopy the genetic interaction of E(spl)D with Notchspl. Although E(spl)-C genes are expressed in temporally and spatially specific patterns, K box-mediated regulation is ubiquitous, implying that other targets of this activity may exist. In support of this, sequence analyses are presented that implicate genes of the Iroquois Complex (Iro-C) and engrailed as additional targets of K box-mediated regulation (Lai, 1998b).

Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation

Micro RNAs are a large family of noncoding RNAs of 21-22 nucleotides whose functions are generally unknown. A large subset of Drosophila RNAs has been shown to be perfectly complementary to several classes of sequence motif previously demonstrated to mediate negative post-transcriptional regulation. These findings suggest a more general role for micro RNAs in gene regulation through the formation of RNA duplexes (Lai, 2002).

A new strategy of gene regulation was defined by the activities of Caenorhabditis elegans let-7 and lin-4. These RNA molecules of 21-22 nt are complementary to the 3' untranslated regions (UTRs) of target transcripts and mediate negative post-transcriptional regulation through RNA duplex formation. Several recent reports now reveal that a large family of RNAs of 21-22 nt, collectively termed micro RNAs (miRNAs), exists in organisms as diverse as worms, flies and humans. Although it was presumed that many of these new miRNAs would also act in post-transcriptional gene regulation, initial searches did not reveal obvious targets based on sequence complementarity (Lai, 2002).

In Drosophila, two 3'-UTR sequence motifs, the K box (cUGUGAUa) and the Brd box (AGCUUUA) mediate negative post-transcriptional regulation. Although originally identified in the 3' UTRs of Notch pathway target genes encoding basic helix-loop-helix (bHLH) repressors and Bearded family proteins, modes of regulation mediated by both motifs are spatially and temporally ubiquitous. This suggests that at least some of the many other Drosophila transcripts that contain K boxes or Brd boxes in their 3' UTRs are also actively regulated by these motifs. Since RNA-binding proteins typically show relatively relaxed binding specificities, it was hypothesized that an RNA component might be involved in recognition of these highly constrained motifs. This was bolstered by the finding that another motif common to the 3' UTRs of many of the same Notch pathway target genes, the GY box (uGUCUUCC), is complementary to and mediates RNA duplex formation through the proneural box (AUGGAAGACAAU), a motif located in the 3' UTRs of transcripts encoding proneural bHLH activators, (Lai, 2002).

Drosophila miRNAs encoded by 11 of 21 distinct genomic miRNA loci are complementary to the K box at their 5' end, with all but miR-11 having a perfect (8/8) antisense match to the extended K box consensus (UAUCACAG). Notably, the most 5' nucleotide of miR-11 is a cytosine residue, making it complementary to the second most common nucleotide at this position in identified K boxes. In addition, perfect antisense matches to the Brd box and GY box were found at the 5' ends of fly miR-4 and fly miR-7, respectively. The precise complementarity of these miRNAs to K box, Brd box and GY box motifs suggests that they bind these sequences in 3' UTRs and, in the case of the former two motifs, mediate negative post-transcriptional regulation. Complementarity between miRNAs and 3' UTRs extends beyond core sequence motifs in many cases, providing additional support for the existence of the proposed RNA duplexes. Examples exist of extended miRNA complementarity to 3' UTRs containing K boxes, Brd boxes and GY boxes. Complements to all three sequence motifs are located exclusively at the 5' ends of miRNA, suggesting that some aspect of regulation may be shared by these different miRNAs. For example, a common factor might be involved in the recognition or stabilization of these short miRNA-3' UTR duplexes (Lai, 2002).

Several miRNAs complementary to K boxes (miR-11 and the miR-2b and miR-13 subfamilies) are broadly expressed throughout Drosophila development, consistent with their proposed involvement in temporally ubiquitous regulation mediated by K boxes; the GY box-complementary miRNA miR-7 is similarly broadly expressed during development. The expression of the single identified Brd box-complementary miRNA miR-4 is restricted to embryogenesis. However, since the search for miRNAs has not yet been saturating, other miRNAs complementary to Brd boxes that are expressed later in development might yet be found (Lai, 2002).

The regulatory role of the K box and Brd box in other organisms has not yet been tested. Nevertheless, the presence of their complements in worm and human miRNAs suggests that these modes of regulation have potentially been conserved. Notably, the complements to these motifs are also located specifically at the 5' ends of miRNA. The restricted location of complements in these different species further suggests that the regulatory targets of many other miRNAs will be determined by the sequence of their 5' ends. In agreement with this idea, most of the known lin-4 and let-7 target sequences also involve perfect complements with the 5' ends of these miRNAs. Systematic searches for the complements of other 5' miRNA ends in 3' UTRs may therefore identify new post-transcriptional regulatory sequence elements. It should be noted, however, that despite the existence of three conserved sites in the lin-14 3' UTR that include perfect complements to lin-4, normal regulation of lin-14 actually depends on variant lin-4 binding sites containing a bulged nucleotide in the 5' complementary region. Thus, this rule is probably not absolute (Lai, 2002).

Initially, miRNAs are transcribed as RNAs of approximately 70 nt containing a stem-loop structure; these are cleaved by the RNAse III enzyme Dicer to generate the mature miRNA. Curiously, only a single strand of the duplex precursor stem structure is generally stable and is recovered as miRNA. The model proposed here may help to explain this phenomenon, since the strand that is complementary to these identified 3' UTR motifs is nearly exclusively the one that is isolated as miRNA. The single exception is miR-5, whose sequence contains a K box. Notably, miR-5 and the K box-complementary miRNAs miR-6-1,2,3 (whose loci are incidentally located next to each other in the genome) are complementary at 20 of 21 continuous nucleotide positions. This suggests that miR-5 might influence or possibly interfere with the ability of miR-6-1,2,3 to interact with 3' UTRs that contain K boxes (Lai, 2002).

Negative regulation by K box- and Brd box-complementary miRNA must differ from lin-4-mediated regulation, because K boxes and Brd boxes have significant, though distinct, effects on both transcript stability and translational efficiency, whereas lin-4 is thought to act at a step following translational initiation. The GY box does not seem to have a strong effect at the cis-regulatory level. Other miRNAs may show additional regulatory capacities; efforts are underway to understand the different molecular mechanisms of regulation mediated by miRNA-3' UTR RNA duplexes (Lai, 2002).

Protein Interactions

The possibility of protein-protein interactions between m4/alpha and a number of potential cytoplasmic partners was tested using the yeast two-hybrid system. Both Hairless and Su(H) are found in both nucleus and the cytoplasm, making such interactions feasible in vivo, yet neither one is able to interact with either m4 or malpha in yeast. No interactions were observed, not with an intracellular fragment of Notch, NICN1, nor with the cytoplasmic factor Dx. Another possibility tested was whether m4/alpha might interact with one another or with Brd. Brd LOF alleles do not modify the overexpression phenotype of m4/alpha. Furthermore, there is no synergy in bristle induction when m4 is co-expressed with malpha; rather the number of ectopic bristles is the same as with two copies of UAS-m4. Consistent with this is the inability of m4 and malpha to homo- or hetero-dimerize among themselves in the two-hybrid assay. Therefore, there is no genetic or molecular evidence for interactions among M4, Malpha and Brd (Apidianakis, 1999).


DEVELOPMENTAL BIOLOGY

Cell-cell signaling through the Notch receptor is a principal mechanism underlying cell fate specification in a variety of developmental processes in metazoans, such as neurogenesis. An investigation is described of seven members of a novel gene family in Drosophila with important connections to Notch signaling. These genes all encode small proteins containing predicted basic amphipathic alpha-helical domains in their amino-terminal regions, as described originally for Bearded; accordingly, they are referred to as Bearded family genes. Five members of the Bearded family are located in a newly discovered gene complex, the Bearded Complex; two others reside in the previously identified Enhancer of split Complex. All members of this family contain, in their proximal upstream regions, at least one high-affinity binding site for the Notch-activated transcription factor Suppressor of Hairless, suggesting that all are directly regulated by the Notch pathway. Consistent with this, it has been shown that Bearded family genes are expressed in a variety of territories in imaginal tissue that correspond to sites of active Notch signaling. Overexpression of any family member antagonizes the activity of the Notch pathway in multiple cell fate decisions during adult sensory organ development. These results suggest that Bearded family genes encode a novel class of effectors or modulators of Notch signaling (Lai, 2000).

Overexpression of Brd causes adult phenotypes closely resembling those conferred by loss-of-function mutations in N pathway genes. These phenotypes include both bristle multiplication and bristle loss; the former is due to the specification of supernumerary SOPs, while the latter is caused by inappropriate allocation of cell fates within the bristle lineage. Likewise, overexpression of other Brd family genes similarly interferes with cell fate specification events controlled by N pathway activity. By comparison with the results with Brd, m4, and malpha, overexpression of Bob and Tom each cause much stronger mutant phenotypes. With one copy of UAS-Bob, a strong tufting or lethal phenotype is observed, with bristle tufting extending to most macrochaetes and microchaetes, as well as occasional bristle loss. UAS-Tom causes the most severe effects of all Brd family members when expressed under the control of sca-GAL4, with most lines giving high percentages of lethality at late pupal/pharate adult stages. The relatively infrequent escapers typically exhibit strong tufting of nearly all macrochaetes and microchaetes and frequently display some degree of bristle loss, especially on the legs (Lai, 2000).

If the gain-of-function effects reported here are indicative of the normal direction of Brd family protein function, and if all members of the Brd gene family are indeed targets of transcriptional activation by this pathway, as has been hypothesized, then Brd family proteins are excellent candidates to mediate a negative feedback mechanism in N signaling. However, a full understanding of Brd family protein function must ultimately incorporate loss-of-function genetic data, which, owing to apparent functional overlap among these genes, is not available. Thus, it is entirely possible that overexpression of Brd family proteins, rather than reinforcing or exaggerating their wild-type activity, instead causes a 'dominant negative' effect; in this case, these proteins may normally function as positive effectors of N signaling (Lai, 2000).

An important issue concerning the function of Brd family proteins is whether they exert their effects on N signaling on the sending or receiving side of the process, or both. Preliminary evidence suggests that overexpression of Brd family genes is able to exert a cell non-autonomous effect on lateral inhibition in proneural clusters, consistent with the possibility that these proteins can antagonize the ability of a cell to send an inhibitory signal. This is of considerable interest, since relatively little is known about the detailed structure and function of the N pathway upstream of the N receptor (Lai, 2000).

Embryonic

Because of the high degree of structural similarity between malpha and m4, the expression patterns of both genes during embryonic development and in imaginal discs were compared. Embryonic expression patterns are nearly indistinguishable, and appear very similar to those of E(spl) bHLH genes, particularly for m5, m7 and m8. Characteristic for this group of genes is the early mesectodermal expression, which appears shortly before the onset of gastrulation. Later on, transcripts of malpha and m4 are detected in the neuro-ectoderm as well as in the mesoderm in a highly dynamic pattern in many stages of embryogenesis. In imaginal discs, the expression domains of malpha and m4 are similar to those described for different E(spl) bHLH genes. Transcripts of m4 accumulate primarily within presumptive proneural clusters of eye-antennal, wing and leg discs, a pattern remarkably similar to that of m8 or m7 expression. However, malpha transcripts are detected in a pattern matching very closely that of mbeta expression. In the eye disc, malpha is expressed not only within but also posterior to the morphogenetic furrow. In the wing pouch, staining of presumptive intervein regions and wing margin is apparent. In the leg disc as well as in the notal part of the wing disc, a more general expression is observed with highest concentration in areas encompassing proneural clusters. The expression patterns suggest that both genes are under the same regulatory control as are the different E(spl) bHLH genes and thus, might have a role in Notch mediated cell differentiation as well. Surprisingly, m2 transcripts also accumulate in a pattern reminiscent of the transcript distribution of E(spl) bHLH genes although there are no structural similarities with either the bHLH or the m4/malpha genes. At first, m2 transcripts are detected in a very dynamic pattern in the neuro-ectoderm of stage 9 embryos. At stage 10/11 the transcripts accumulate at high levels in the presumptive mesoderm, however, they disappear quickly with the onset of germ band retraction. Imaginal disc expression is rather weak (Wurmbach, 1999).

Larval

In eye disc, m2 transcripts are observed close to as well as posterior to the morphogenetic furrow. In the wing disc, areas of proneural clusters stain weakly, as does the dorso-ventral boundary and vein/intervein regions. These patterns are typical of E(spl) bHLH gene expression. Therefore, m2 appears to be regulated in a similar manner like E(spl) bHLH and m4/malpha genes and could also serve as a Notch target gene (Wurmbach, 1999).

Postembryonic expression patterns of Brd family genes were examined by in situ hybridization. In wing imaginal discs of third-instar larvae, Brd and E(spl)m4 transcripts accumulate specifically in the full complement of sensory organ proneural clusters. Similarly, the complex pattern of E(spl)malpha expression in the wing disc includes proneural clusters, although malpha transcript accumulation in the clusters consistently appears broader and more diffuse than that of Brd or m4. In addition, malpha transcripts appear in a narrow stripe along the dorsoventral boundary of the wing pouch, as well as along wing vein borders. In contrast, neither Bob nor Tom exhibit any patterned expression in the wing disc, although Tom may be generally expressed at a very low level in this tissue. In the eye imaginal disc, four of the five Brd family genes in this study are expressed in the vicinity of the morphogenetic furrow, the exception being Bob, which is not detectably expressed in either the eye or antenna discs. Transcripts from the different Brd family genes accumulate with distinct spatial profiles relative to the morphogenetic furrow. Brd is expressed in two closely spaced stripes, one just anterior to, and one within and posterior to, the dpp furrow stripe. Transcripts from m4, by contrast, appear in a strong band that is largely just anterior to the zone of dpp-lacZ expression. malpha shows expression in a pattern that overlaps, and extends posterior to, the marker stripe. Finally, Tom expression somewhat resembles that of Brd, in that its transcripts accumulate in two stripes lying anterior and posterior to the dpp-lacZ stripe (Lai, 2000).

The patterns of transcript accumulation from these genes were examined during pupal wing development, to assess the possible expression of these transcripts in sensory organ lineages and in the vicinity of the developing wing veins. The members of the Brd-C are not expressed at detectable levels in the pupal wing at 8 hours after puparium formation (APF), although Brd and Tom transcripts are present in the large clusters of proximal campaniform sensilla at this time. By contrast, m4 and malpha in the E(spl)-C display both proximal campaniform expression and specific wing margin expression at 8 hours APF. m4 transcripts accumulate in a set of anterior wing margin cells at this stage. Based on their spacing, they are likely to represent cells in the lineage of the chemosensory organs that appear in dorsal and ventral rows on the margin. Since transcripts from E(spl)mgamma accumulate in these organs, it appears that at least one Brd family member and at least one bHLH gene in the E(spl)-C share this aspect of their expression. It has been found that malpha is expressed at this time (8 hours APF) in a broad domain of wing margin cells that includes cells of the posterior as well as the anterior margin, and also in an incomplete wing vein boundary pattern. This latter observation prompted an examination of the accumulation of malpha transcripts in later pupal wing discs. At 24 hours APF, malpha is indeed expressed in a largely complete pattern consisting of thin rows of cells at all vein/intervein boundaries. This is highly reminiscent of the pattern of transcript accumulation from both Notch and the bHLH gene E(spl)mbeta at approximately the same time. In addition, malpha transcripts remain present throughout the wing margin (both posterior and anterior) at this stage. malpha expression in the pupal wing is highly dynamic, however: by 30 hours APF, its transcripts have nearly gone from the margin, are excluded from vein/intervein borders, and appear instead in the veins themselves and in non-vein wing blade tissue. Taken together, these observations strongly suggest that at least one Brd family member may have a role in wing vein development. In summary, in developing imaginal tissue, Brd family members are expressed specifically in multiple territories in which Notch signaling-dependent cell fate decisions take place (Lai, 2000).

Phenotypic effect of UAS-m4 and UAS-malpha Overexpression

Overexpression UAS-m4 and UAS-malpha causes downregulation of E(spl) bHLH expression accompanied by cell autonomous overcommitment of sensory organ precursors and tufting of bristles. Experiments by Nagel (2000), suggest that the overexpression phenotype of E(spl) m4 and E(spl) malpha obrained by Apidianakis (1999) is likely to be due to a dominant negative effect and does not reflect the biological function of these two genes. The results of and interpretation of Apidianakis (1999) are presented here for a complete record.

Negative regulation of the Notch pathway by overexpression of E(spl) m4 and malpha is specific to the process of sensory organ precursor singularization and does not impinge on other instances of Notch signaling. Identical effects were produced by both UAS-m4 and UAS-malpha transgenes, with quantitative variations among lines attributed to position effects; therefore they are collectively referred to as m4/alpha. Macrochaetae, microchaetae and campaniform sensilla were similarly affected by m4/alpha overexpression. For example, expression of UAS-m4 ahead of the antero-posterior boundary of the wing disk (by ptc-GAL4) produces excess scutellar bristles and anterior cross-vein (ACV) campaniform sensilla (the sensory organs that arise from this region of the disc). Characteristic of the m4/alpha overexpression phenotype is the multiplication of sensory organs at their normal locations; the extra bristles are frequently tufted together. Other than sensillum multiplication, no effects are observed in the wing, where Notch signaling is known to affect wing margin integrity and vein thickness. No wing pattern aberrations are observed when single copy UAS transgenes are driven with GAL4 lines that express predominantly in the wing pouch, such as 32B and ombmd653. This has been confirmed by examining vg(boundary)-lacZ and wg-lacZ in m4/alpha overexpression backgrounds; the patterns of these wing-margin specific markers were identical to wild-type (Apidianakis, 1999).

Overexpression of proneural genes of the bHLH type, like those of the Achaete-Scute complex (see Achaete), also gives rise to extra sensory organs. Yet, the phenotype observed for m4/alpha is quite distinct from that of overexpression of proneural genes. The latter produce ectopic bristles at new locations, such as the wing blade, something never observed with m4/alpha. Also, the ectopic bristles produced by UAS-lethal of scute are spaced, suggesting that they arise from individually spaced SOPs. To compare the SOP pattern produced by UAS-m4/alpha with that of UAS-l'sc, each one was expressed with the ap-GAL4 driver in the background of the SOP specific enhancer trap neurA101. Although l'sc yields a pattern of discrete SOPs randomly dispersed throughout the dorsal half of the wing pouch (the apterous expression domain), m4/alpha gives clustered SOPs at the normal locations where single SOPs would have arisen in the wild-type, as expected from the adult phenotype. Therefore, it appears that m4 and malpha do not have proneural function; rather, they rely upon proneural gene expression to promote the SOP fate. This was confirmed by combining ectopically expressed m4 (ap-GAL4;UAS-m4) with a deficiency for the proneural genes ac and sc, Df(1)sc10-1. Flies hemizygous for sc10-1 have bald nota, as no SOPs are specified in the absence of proneural gene activity. Expression of m4 in this background does not restore any chaetae, suggesting that m4 is unable to induce sensory organs in the absence of proneural proteins (Apidianakis, 1999).

Overexpression of m4/alpha blocks lateral inhibition. The SOP pattern is a result of an interplay between proneural proteins, which promote neural fate, and Notch signaling, which inhibits it. The fact that supernumerary chaetael/SOPs arise in close apposition to each other suggests that the contact-dependent Notch signaling that normally counters SOP fate may be compromised. To locally block lateral inhibition for comparison purposes, a well characterized negative regulator of Notch signaling, Hairless (H) was overexpressed. H is known to negatively modulate Notch signaling by interfering with the activity of the transcriptional activator Su(H), by which at least part of the Notch signal is transduced to the nucleus (Apidianakis, 1999).

Generally, the effect of H overexpression is similar to that of m4/alpha. At the ACV (anterior cross-vein campaniform sensillum), L3 (third longitudinal vein campaniform sensillum) and wing margin clusters, the extent of SOP overcommitment is comparable to that caused by m4/alpha, whereas at the dorsal radius H gives much higher numbers of supernumerary SOPs. The effects of H differ from those of m4/alpha in two further respects. (1) H abolishes some wing margin sensilla, presumably by interfering with the Su(H)-dependent inductive Notch signaling that sets up the dorsoventral boundary, which subsequently induces margin SOPs. m4/alpha does not affect the process of dorsoventral wing patterning, consistent with the presence of a full complement of margin SOPs. (2) In the adult phenotype, whereas m4/alpha produces solely bristle tufting, H overexpression variably produces naked patches or double-shaft socketless bristles, consistent with its proposed role in the SOP lineage cell fate decisions. Ectopic expression of m4/alpha gives neither of these phenotypes, suggesting that it affects only SOP singularization but not the SOP lineage. In order to test this hypothesis, pupal nota were stained with antibodies directed against Elav, a neuron specific marker, and Pros, specific to the sheath cell. There is a one-to-one correspondence between Elav positive and Pros positive cells. Therefore, overexpression of m4/alpha does not upset the Notch/Numb mediated asymmetric divisions in the SOP lineage. The only step in sensory organ development that m4/alpha seem to affect is that of lateral inhibition, which restricts the number of SOPs produced per proneural cluster (Apidianakis, 1999).

As it is likely that m4/alpha affect a pathway of cell-cell communication, it is important to determine whether they do so by interfering with signal emission or with signal reception. In the former case, their effect would be non cell-autonomous. To test this, UAS-m4 was overexpressed in clones of cells. Patches overexpressing m4 give the expected phenotype of microchaeta tufting marked with f, a marker carried by the Ubx transgene, whereas adjacent non-expressing f1 bristles are always single. Moreover, f marked bristles at clone boundaries are usually multiplied, that is, they are not 'rescued' by their proximity to wild-type tissue. It cannot be concluded that this autonomy holds down to the single cell level: the epidermal cell phenotypes could not be scored. Still, it is tentatively concluded that the effect of m4 is achieved through blocking of signal reception (Apidianakis, 1999).

How do m4/alpha act to negatively modulate lateral inhibition? This new family of proteins contains no known structural motifs that would point toward a possible function. To study the level of functioning of the Notch pathway a number of molecular markers have been used. One indicator of Notch signaling is the expression of the E(spl) bHLH genes, a subset of which are recognized by the monoclonal antibody 323. Wild type proneural clusters are positive for mAb323 immunoreactivity with the exception of single cells, which represent the committed SOPs. Unlike severe Notch loss of function, which abolishes mAb323 immunoreactivity, overexpression of m4/alpha causes a milder overall loss of staining with a subset of cells within the proneural cluster displaying undetectable levels of mAB323. The negative cells are always in contact, surrounded by E(spl) positive cells. Most likely, these cells correspond to the clustered neurA101 positive SOPs, since loss of E(spl)bHLH expression favors the SOP fate (Apidianakis, 1999).

Downregulation of the E(spl)bHLH protein levels by m4/alpha could be at the level of transcription or post-transcriptional. To test this, E(spl) derived reporter genes were used in both the eye and in a tissue-culture assay. In the eye, m8-lacZ was used. This drives expression in a subset of proneural clusters. m4 was overexpressed using the omb-GAL4 driver, which expresses in a broad central domain of the wing pouch, and apmd544-GAL4, which expresses in the dorsal compartment of the wing disc. In both cases a dramatic reduction of m8-lacZ activity was observed within the m4 overexpression domain. This occurs even in regions where no ectopic sensory organs are formed, such as the posterior wing margin, suggesting that the loss of m8-lacZ staining is not simply a consequence of overcommitment of SOPs. The effects observed with the 323 antibody are not identical to those seen by X-gal staining, the latter displaying a spatially more uniform reduction in staining as a result of m4 overexpression. The difference may be attributed to the different sensitivity of the techniques used, or to the fact that the two experiments assay the expression of different genes [mAb323 does not recognize E(spl)m8]. Further studies are needed to determine if m4/alpha affect the expression of different E(spl)bHLH genes differentially; the conclusion at present is that overexpression of m4/alpha decreases E(spl)bHLH protein levels, at least partly through blocking transcription (Apidianakis, 1999).

There are a number of Notch pathway components that m4/alpha could interact with to downregulate the expression of E(spl)bHLH genes. In an exploratory experiment, m4 or malpha were over-expressed in genetic backgrounds heterozygous for various Notch pathway mutations to detect possible genetic interactions. Of the mutations tested, five modify the phenotype: N264-40 and E(spl) b32.2 increase the number of supernumerary bristles, and N Ax, Dp(1;2)N+ and H2 decrease it. The effects of N alleles agree with the proposed negative regulation of Notch signaling by m4/alpha. The further suppression of lateral inhibition by halving the dose of E(spl) is also not surprising, given the data that m4/alpha acts by downregulating the transcription of E(spl)bHLH genes. The suppression of supernumerary bristles by a reduction in H can finally be accounted for by increased transcriptional activation of the E(spl)bHLH genes, since H normally blocks the activity of the Su(H) transcriptional activator. Instances of severe H LOF (H1/H2) or strong N GOF (NAxM1/Y) produce nota that are essentially bald. In these backgrounds m4/alpha overexpression appears unable to induce bristles or SOPs (as revealed by anti-Asense staining) (Apidianakis, 1999).

Whereas the genes encoding m4 and malpha are located within the E(spl) region at 96F, two other members, Brd and a newly identifed EST transcript, reside in 71A. m4, malpha and Brd, are normally expressed in proneural clusters from which individual SOPs will arise, and m4/alpha (but not Brd) are transcriptionally induced by Notch signaling. The expression pattern and Notch-dependence of the fourth member is still unknown. Loss of function of either Brd or m4 gives a wild-type phenotype (Leviten, 1996 and Apidianakis, 1999), pointing to functional redundancy within this protein family. GOF phenotypes of Brd and m4/alpha are also virtually identical: they produce supernumerary SOPs. The only known difference to date is slight: m4/alpha seem unable to affect cell fates in the sensory organ lineage, whereas strong Brd gof occasionally causes naked cuticle patches due to pIIa transformation to the pIIb fate. Based on the similarity of both LOF and GOF phenotypes, a model is suggested whereby all members of this family have a similar mode of action, namely to antagonize Notch. Their action (at least in the case of m4 and malpha) can be accounted for by reduced expression of E(spl)bHLH proteins, which are well known effectors of Notch signaling and antagonists of the SOP fate. Despite functional similarity, sequence similarity is not high between Brd and the other members: most importantly, Brd lacks the well conserved C-terminal domain present in all other members. What might the role of this domain be? One possibility is that it constitutes a regulatory domain whose function becomes dispensable upon overexpression of the protein (Apidianakis, 1999).

The fact that m4/alpha are turned on by Notch signaling, even though they act against the downstream implementation of the signal, might be counter-intuitive, yet is not unprecedented. Other signaling pathways employ similar mechanisms of negative autoregulation. For example, activation of the EGF receptor results in expression of argos, which encodes an inhibitory ligand of Egfr. Another example is the Hh pathway, where signaling upregulates expression of patched, coding for a transmembrane receptor of Hh, which inhibits signal transduction. A major difference, however, is that in both examples, inhibition serves to spatially restrict the effects of signaling, and, in the case of Argos, is not cell autonomous. In contrast, m4/malpha cells act autonomously, antagonizing the Notch signal within the same cell that should be responding to it. In fact both signaling mediators (E(spl)bHLH proteins) and antagonists (m4/alpha) are turned on by Notch signaling, via the same mechanism, namely Su(H)-dependent transcriptional activation. One possible function of this apparently conficting co-expression is to set a threshold for the level of Notch signaling needed to divert a cell from the SOP fate. An alternative (and not exclusive) possibility might be that these factors ensure that the N effect is very transient. In this respect, it is worth noting that all Notch-responsive genes within the E(spl) locus, both the bHLH and m4/alpha, are short and intron-less, ensuring rapid accumulation of their products. Their degradation is rapid, too: their transcripts contain special destabilizing signals, and the same likely holds true for their protein products as well. These attributes make the presence of these Notch responsive factors very transient and dynamic, such that small differences in temporal accumulation might be hard to detect. For example, members of the m4/alpha family can be thought of as factors that are activated slightly later than E(spl) bHLH to switch off a round of Notch signaling, after the positive mediators [E(spl) bHLH proteins] have accomplished their task. To address such a function, detailed studies are needed, which will focus on the precise temporal sequence of E(spl)bHLH versus m4/alpha expression (Apidianakis, 1999).

What is more unexpected in these findings is the high specificity of m4/alpha for the process of SOP singularization and the apparent indifference of other Notch mediated processes to m4/alpha overexpression. Overexpression of Hairless or Numb, two other well characterized Notch pathway inhibitors, affects a much broader range of developmental processes, e.g. cell fates in the SOP lineage, wing margin formation, and wing pro-vein restriction. Hairless may normally participate in all of these events. In contrast, Numb is specific for the asymmetric divisions in the SOP lineage, but can mildly affect other processes when ectopically expressed. Could the inability to detect phenotypes in other Notch-dependent processes be simply due to low levels of transgene expression? This is believed not to be the case, since a large number of GAL4 drivers were utilized that otherwise give SOP phenotypes with high expressivity and penetrance. A possible explanation for the refractoriness of other processes to m4/alpha expression is that these proteins are subject to post-translational regulation that masks their activity in most cell types. One type of such regulation might be the association with an essential co-factor, whose expression could be restricted to the proneural cells. Yet, two-hybrid analysis fails to reveal such a co-factor among the various likely candidates tested. The one lead regarding the molecular mode of action of m4/alpha is the documented downregulation of E(spl)bHLH gene expression. Since these are target genes of Notch, such an effect could result from a block in any of the steps involved in Notch signal transduction. The tissue culture results suggest that m4/alpha can block E(spl)bHLH genes even when activated Notch is exogenously provided, suggesting that these factors act at a step after Notch activation. However, the effects observed in these experiments are rather modest compared to the in vivo effects and thus it is uncertain that they reflect the same activity of the m4/alpha molecules (Apidianakis, 1999).


EFFECTS OF MUTATION

The Enhancer of split complex [E(spl)-C] of Drosophila is located in the 96F region of the third chromosome and comprises at least seven structurally related genes: HLH-m delta, HLH-m gamma, HLH-m beta, HLH-m3, HLH-m5, HLH-m7 and E(spl). The functions of these genes are required during early neurogenesis to give neuroectodermal cells access to the epidermal pathway of development. Another gene in the 96F region, namely groucho, is also required for this process. However, groucho is not structurally related to, and appears to act independently of, the genes of the E(spl)-C; the possibility is discussed that groucho acts upstream of the E(spl)-C genes. Indirect evidence suggests that a neighboring transcription unit (m4) may also take part in the process. Of all these genes, only gro is essential; m4 is a dispensable gene, the deletion of which does not produce detectable morphogenetic abnormalities, and the genes of the E(spl)-C are to some extent redundant and can partially substitute for one another. This redundancy is probably due to the fact that the seven genes of the E(spl)-C encode highly conserved putative DNA-binding proteins of the bHLH family. The genes of the complex are interspersed among other genes that appear to be unrelated to the neuroepidermal lineage dichotomy (Schrons, 1992).


EVOLUTIONARY HOMOLOGS

m4 and malpha are both members of the same large family of proteins. To identify proteins structurally similar to m4/alpha, BLAST analysis was performed against all available protein and nucleic acid data bases. The highest scoring hits were consistently two Drosophila ESTs: AA246754 and AA433222. When the two were analyzed against each other, it became clear that they overlap, thus defining a single genetic locus. The predicted protein product from the assembled sequence shows similarity across the whole polypeptide chain to both m4 and malpha. m4-ESTs are 31% identical (48% similar) and malpha-ESTs are 31% identical (55% similar). The genomic cosmid library of the European Drosophila Genome Project was screened using a PCR product from this EST and two positive clones were found. Both of these map to 71A (I. Siden-Kiamos, personal communication to Apidianakis, 1999), a region away from the E(spl) Complex (96F), but close to the Brd locus (whose product bears 22% identity, 46% similarity to the N-terminal half of EST), making this a dispersed gene family. Besides their protein product similarity, m4 and Brd have been reported to share common sequence motifs in their 30 untranslated regions (UTRs), the Bearded box, the K box and the GY box, all of which are interestingly also found in E(spl)bHLH transcripts. Two each of Brd- and K- boxes were found, but no GY box in malpha, whereas the EST transcript reveals all three motifs. The Brd- and K-boxes have been shown to confer mRNA destabilization. Quick RNA (and protein) turnover is desirable for factors that are deployed transiently in response to intercellular signals and whose effect, therefore, has to be downregulated when the signaling is turned off. The function of the third 30 UTR motif, the GY-box, is presently not understood. Interestingly, no sequences from other species gave high BLAST scores in searches, thus presently limiting the members of this family to Drosophila (Apidianakis, 1999).

Brd and most genes of the E(spl)-C (including both bHLH genes and m4) are also subject to common modes of negative post-transcriptional regulation via defined sequence motifs present in their 3' UTRs. In particular, K boxes (TGTGAT) and Brd boxes (AGCTTTA), which are broadly distributed within the 3' UTRs of these genes, mediate negative regulation of transcript accumulation and translational efficiency. Two Brd boxes and two K boxes have been identified in the 3' UTR of malpha, a K box and a Brd box in the 3' UTR of Tom, and two K boxes in the 3' UTR of Bob. Moreover, the second K box in Bob is directly adjacent to a CAAC motif, a sequence that has been implicated in augmentation of regulation by an associated K box. Bob's 3' UTR does not contain a canonical Brd box, but does contain a 7/7 match to a variant of the Brd box (TGCTTTA) found in the D. hydei ortholog of E(spl)m4. Overall, the presence of canonical K box and Brd box sequences in the 3' UTRs of Bob, Tom and malpha strongly suggests that most genes of the Brd-C and E(spl)-C are subject to the same two modes of negative post-transcriptional regulation (Lai, 2000).

A third class of conserved 3' UTR sequence motif, the GY box (GTCTTCC), is also shared by Brd and genes of the E(spl)-C. Although the precise function of this motif is poorly understood, it is possible that this motif has a role in forming RNA:RNA duplexes with a complementary sequence motif (the proneural box, AATGGAAGACAAT) found in the 3' UTRs of proneural genes, including ac, lethal of scute. and atonal (ato). The 3' UTRs of both Bob and Tom each contain a pair of GY boxes. Closer examination of the GY boxes of Bob, Tom and Brd reveals an unexpected degree of sequence identity in the nucleotides flanking the GY box heptamer in Brd-C genes. The GY boxes of Tom are found within a 19/19 direct repeat in the Tom 3' UTR, while Bob's GY boxes fall within a 15/15 direct repeat in its 3' UTR. Moreover, an exact 16-bp sequence including a GY box is common to the 3' UTRs of Brd, Bob, and Tom, and all five GY boxes in these Brd-C genes are contained within an exact 15/15 identity. It is striking that this latter sequence is exactly complementary to a 15-nt sequence shared by proneural boxes located in the ato and l'sc 3' UTRs. That the GY boxes of all Brd-C genes should share such an exceptional relationship with the proneural boxes of divergent proneural genes located on different chromosomes (ato and l'sc) strongly suggests that these complementary sequence elements are subject to common constraint. The two GY boxes in the 3' UTR of E(spl)m4 are more related to the extended GY box consensus just described than are the GY boxes of most E(spl)-C bHLH transcripts. Thus, the constraint on m4's GY boxes similarly appears to extend well beyond the core seven nucleotides of this motif, in a way that is also evidently connected to the proneural box sequence. Finally, the 3' UTR segments containing the second GY box of Bob and the first GY box of Tom are related by an extraordinary 32-nt exact identity. That members of distinct subfamilies of the Brd gene family should share such an extended GY box-containing identity further underscores the sequence constraint associated with this motif, and may suggest the existence of a common 'partner' gene for Bob and Tom that carries a complementary sequence. The complementary 3' UTR sequence motifs found in proneural genes and Brd family genes can mediate the formation of RNA:RNA duplexes in vitro. Since transcripts of members of the proneural gene family and the Brd gene family co-accumulate in all developmental settings where neurogenesis occurs, it is suggested that these RNA:RNA duplexes also form in vivo, although the possible regulatory consequences of this association remain to be determined (Lai, 2000).

The sequences of Drosophila ESTs encoding apparent paralogs of both Brd and E(spl)m4 have been deposited in the GenBank database. PCR products containing these EST sequences were used as probes to isolate full-length cDNA and genomic DNA clones for both genes, which have been named Bob ( Brother of Brd) and Tom (Twin of m4), respectively. In addition, genomic DNA has been cloned and sequenced that includes the previously identified E(spl)malpha locus: its predicted protein product is also strongly related to that of E(spl)m4. The predicted amino acid sequences of what is referred to as Brd family proteins can be classified as Brd-like (Brd and Bob) or m4-like (m4, malpha, and Tom), based on their relative sizes and degree of amino acid similarity. Although there are a few well-conserved regions in these proteins, particularly within the C-terminal half of the longer m4-like proteins, it is obvious that Brd family members are not in general highly related at the primary structure level. Brd and m4 are related by secondary structure, since a domain located near the N-terminus in both proteins is predicted to form a basic amphipathic helix. Similar N-terminal domains found in Bob and E(spl)malpha are likewise strongly predicted to form basic amphipathic helices, while a proline residue in the center of the corresponding region of Tom suggests that its 'helix' may be kinked or separated into two helices. The strong basic amphipathic character of these N-terminal domains of Brd family proteins may be considered a defining structural feature. Brd family proteins also share certain classes of consensus phosphorylation sites, namely protein kinase C (PKC) sites in their N-terminal regions and casein kinase II (CK II) sites in their C-terminal portions. The similar placement of these consensus sites in the context of otherwise weakly related amino acid sequences suggests that they may be relevant for the regulation of Brd family protein function (Lai, 2000).

Bob and Tom genes were localized to the Drosophila genome physical map using a filter grid library of P1 clones. Interestingly, Bob and Tom map to a set of P1 clones covering cytological region 71A1-2, the known chromosomal location of Brd. Characterization of this genomic region shows that Tom gene is found to lie only about 2 kb upstream of the previously described Brd transcription unit, with Bob about 20 kb upstream of Tom. Surprisingly, the genomic DNA corresponding to the Bob EST and cDNA clones is triplicated, such that three distinct, tandemly arranged genomic loci have the capacity to encode nearly identical Bob transcripts. This triplicated structure has been observed in two independent, overlapping lambda phage genomic DNA clones. Significantly, small sequence differences in the transcribed portions of the different Bob genomic loci are also represented in the cDNA clones, allowing for the conclusion that at least two copies are transcriptionally active in wild-type flies. The three gene copies have been designated Bob A, B, and C, the encoded transcripts and proteins are referred collectively as 'Bob'. Thus, a minimum of five Brd family genes are contained within an approximately 30-kb interval that is referred to as the Brd Complex (Brd-C), and this complex contains both Brd-like (Brd and Bob) and m4-like (Tom) genes. The E(spl)-C contains at least two m4-like genes, m4 and malpha. Promoter regions of Brd family genes within the Brd-C and E(spl)-C contain consensus binding sites for proneural proteins and for Suppressor of Hairless (Lai, 2000).

The commonality of an N-terminal basic domain in all Brd family proteins, along with their similar phenotypic effects when over- or mis-expressed, suggests that they may have a common biochemical mechanism of action and may interact with a common target or targets. The conserved C-terminal extension found in the m4-related proteins (m4, ma, and Tom) further suggests that this subfamily may have additional functions that are not shared by the shorter Brd-related proteins (Brd and Bob). In particular, it is possible that the terminal DRWV/AQA motif in these proteins, by analogy with the conserved C-terminal domain of the E(spl)-C bHLH proteins (which recruits the co-repressor Groucho), may also mediate protein-protein interactions. A similar possibility exists for the shared PVXFXRTXXGTFFWT motif (Lai, 2000).

Barbu (Bbu), an alternative name applied to Twin of m4 (Tom), can antagonize Notch signaling activity during Drosophila development. In this study Barbu/Tom was isolated in a search for proteins that physically interact with Tinman. As explained below, it is uncertain whether this physical interaction is of biological significance, but it is clear that this gene functions to antagonize Notch signaling, as does E(spl) region transcript m4,. Ectopic expression studies with Barbu provide evidence that Barbu can antagonize Notch during lateral inhibition processes in the embryonic mesoderm, sensory organ specification in imaginal discs and cell type specification in developing ommatidia. Barbu loss-of-function mutations cause lethality and disrupt the establishment of planar polarity and photoreceptor specification in eye imaginal discs, which may also be a consequence of altered Notch signaling activities. Furthermore, in the embryonic neuroectoderm, Barbu expression is inducible by activated Notch. Taken together, it is proposed that Barbu functions in a negative feed-back loop, which may be important for the accurate adjustment of Notch signaling activity and the extinction of Notch activity between successive rounds of signaling events (Zaffran, 2000).

The temporal and spatial pattern of BBU/TOM mRNA was determined by Northern blot and whole-mount in situ hybridizations. A single 1.3 kb BBU transcript appears at 2-4 hours of development with a peak expression at 4-8 hours after fertilization. BBU mRNA expression decreases dramatically following late embryonic stages until its expression becomes barely detectable after the first instar larval stage. Whole-mount in situ hybridization using the Bbu cDNA insert as a probe first detects Bbu expression in the anterior-most and in central regions of early blastoderm embryos. At the end of the blastoderm stage, Bbu is uniformly expressed, but expression is excluded from the ventralmost part of the embryo, which corresponds to the presumptive mesoderm domain. Because the zinc finger protein Snail is known to act as a repressor within the mesodermal territory, a test was performed to see whether Bbu expression is negatively regulated by snail. Indeed Bbu is ectopically expressed in the presumptive mesoderm in snail mutants. Later, however, shortly after gastrulation and until the end of the germband elongation, Bbu expression is seen throughout the mesoderm. Bbu and Tin co-localize during stages 8 and 9 in the entire mesoderm and during stage 10 in most of the dorsal mesodermal cells. During late germband elongation, when the specification of mesodermal tissues and muscle progenitors initiates, Bbu expression assumes a segmental pattern in the ventral mesoderm. At the same time, Bbu expression in the ectoderm becomes patchy and expression levels increase in the tracheal pit territories. BBU mRNA expression in the ectodermal layer was further studied using confocal microscopy. During germband elongation and at the beginning of neuroblast segregation, BBU mRNA is excluded from one or two rows of ventral ectodermal and mesectodermal cells in each segment. Slightly later, rapid and complex changes in the Bbu expression pattern occur, during which Bbu expression becomes transiently excluded from segmental, bilaterally symmetric patches of cells. Furthermore, at the beginning of germband retraction when most neuroblasts have segregated, Bbu becomes more weakly expressed in the ventral region of the ectoderm as compared to its dorsal expression. During germband retraction, Bbu expression rapidly decays and, after dorsal closure, is only seen in the pair of lymph glands close to the anteriormost portion of the dorsal vessel. An antibody to Bbu protein reveals that Bbu protein expression matches that of BBU mRNA expression (Zaffran, 2000).

In the neuroectoderm, Bbu expression is excluded from the proneural clusters and neuroblasts in which these proneural proteins are expressed. In clusters from which single cells have been selected to maintain proneural gene expression, all cells except for the presumptive neuroblast express Barbu mRNA. These observations raised the possibility that the Notch signaling pathway, while repressing proneural genes, may activate Bbu in the presumptive non-neuroblast cells. Ectopic expression of activated Notch results in the overexpression of BBU mRNA. The presence of three putative Su(H) binding sites in the presumed Bbu promoter sequence indicates that activation of Bbu by Notch is possibly direct. One of these sequences ([-791]CGTGGG-AAA) matches exactly the previously reported consensus sequence for Su(H) sites [(C/T)GTG(G/A)GAA(C/A)]. Thus, the control of Bbu expression with respect to its dynamic 'filling in' of proneural clusters and exclusion from prospective neuroblasts seems to be a direct result of its activation by the Notch signaling pathway in the neuroectoderm. Additional experiments show that overepxression of Bbu in the mesoderm antagonizes Notch signaling giving rise to an increased number of Even-skipped expressing heart cells. Overexpression of Bbu also affects the development of lateral and ventral muscle progenitor muscles. Likewise, overexpression of Bbu increases the number of bristles in the adult (Zaffran, 2000).

Bbu mutants were induced by P-element excision. Although the presumed null allele Bbu99 confers embryonic lethality, no specific abnormalities could be detected in ectodermally or mesodermally derived tissues in homozygous embryos for this mutation. However, the phenotypic analysis of the hypomorphic alleles Bbu8 and Bbu105 proved to be informative. Both Bbu8 and Bbu105 confer pupal lethality and pharate adults homozygous for these mutations fail to undergo head eversion. Bbu hypomorphic alleles rescues the phenotype of homozygous Su(H) mutants, which are larval lethal with small eye discs. Bbu;Su(H) double mutants develop into the pupal stage with pharate adults displaying partially everted heads. Thus, reduced levels of Notch/Su(H) signaling can partially rescue the pupal phenotype caused by reduced levels of Bbu expression. Bbu105 gives rise to a small number of escapers when the flies are grown at low density. The bristles on the notum of these adults, particularly in the anterior portion, show defects in their polarity. Since both planar polarity and lateral inhibition involve the Notch signaling pathway, these results suggest that Bbu may act to adjust the levels of Notch signaling during these processes (Zaffran, 2000).

During third larval instar, Barbu expression occurs in eye-antennal imaginal discs and is restricted to narrow bands of cells on either side of the morphogenetic furrow from which most cells enter the neuronal pathway. Notch signaling has been shown to participate in the control of the expression of the proneural protein Atonal (Ato), which is essential for cell fate specification of the first neuronal cells (photoreceptors 8: R8). In, Bbu105 the pattern of Ato expression appears irregular compared with the wild-type pattern and, occasionally, Ato-stained R8 cells are missing. In addition, Ato levels anterior to the morphogenetic furrow appear increased relative to the levels in the R8 cells. Despite these abnormalities, no obvious defects are detected in the expression of Elav protein, showing that, in most ommatidia, eight neuronal photoreceptor cells are formed. Adult escapers that are homozygous for Bbu105 display rough eye phenotypes. Scanning electron microscopic analysis reveals that the roughened appearance in the eyes of Bbu105 escapers is due to the failure to form a regular array of hexagonal corneal lenses, which is accompanied by an irregular distribution of interommatidial bristles. This phenotype suggests that the Bbu105 mutation causes polarity defects in the ommatidia, which is further confirmed by the analysis of tangential sections through the eyes. The degree of rotation of the ommatidia from homozygous Bbu105 flies appears random since some ommatidia have not rotated at all, while others have rotated only 45°or have continued the rotation beyond the normal 90°. Similar phenotypes also occur with null mutations in frizzled and in flies in which Notch activity is altered during photoreceptor development. Induction of Bbu null mutant clones in the eye results in a severe roughening as well as a size reduction of the ventral half of the eyes containing these large Bbu minus clones. The defects include aberrant rotation of some ommatidia from mutant clones. However, many ommatidia from Bbu minus clones display stronger defects and show a significant reduction of the number of photoreceptor cells to four, five or six cells per ommatidium in the plane of sectioning. Thus, in addition to ommatidial polarity, complete loss of Bbu activity appears to disrupt photoreceptor recruitment. Ectopic expression of Bbu in the eye gives rise to ommatidia that lack a regular organization. Ectopic Bbu gives rise to defects in the corneal lenses, which appear to contain extra sockets, suggesting transformations of photoreceptors to non-neuronal fates. In agreement with this interpretation, tangential sections of such eyes with ectopic Bbu expression show many ommatidia with less than the normal number of photoreceptors. In addition, ommatidia with a full complement of photoreceptors show defects in their chirality. Thus, overexpression of Bbu affects not only the chirality, but frequently also the specification of neuronal cells of the ommatidia (Zaffran, 2000).

Bbu is the first member of this gene family to show a loss-of-function phenotype. While Bbu loss-of-function alleles cause lethality and developmental defects, null mutants for Brd and E(spl) m4 are homozygously viable and do not exhibit any detectable abnormalities. It is likely that the wild-type phenotypes of Brd and m4 null mutants are due to functional redundancy among genes of this family. Partial functional redundancy may also explain the failure to detect any phenotypes in Bbu null mutant embryos. Nevertheless, the observed pupal and adult phenotypes, as well as the genetic interactions of Bbu loss-of-function mutations with Su(H) mutations, are compatible with a Bbu role in the downregulation of Notch signaling activity. Prominent features that are shared with Notch gain-of-function phenotypes are polarity defects and disrupted cell specification during eye development. Similar to heat-shock induction of Nintra, reduction of Bbu activity results in increased levels of atonal expression anterior to the morphogenetic furrow and, in some instances, loss of ato expression in the prospective photoreceptor R8. No overt R3 to R4 transformations are seen in the mis-oriented ommatidia upon reduction of Bbu activity, although such cell fate transformations are observed upon ectopic Notch activation and thought to be a cause for polarity defects. Rather, randomly oriented ommatidia are observed with apparently normally specified photoreceptors, which is very similar to the defects reported for mutations in frizzled and its downstream effector gene dishevelled. Recent models for the origin of the ommatidial polarity have proposed the occurrence of reciprocal Notch/Delta signaling between the prospective R3 and R4 cells. This mutual interaction is thought to be biased by increased Frizzled/Dishevelled activities in R3, which downregulates Notch activity and upregulates the production of Delta in this cell. The observed Bbu mutant phenotype in ommatidia suggests that, upon the reduction of Bbu levels, this bias is removed and the decisions between R3 and R4 fates within each ommatidum become randomized. Thus, it is propose that, in the normal situation, Bbu is required together with activated Frizzled to antagonize Notch activity in the prospective R3 photoreceptor. While a model is preferred in which Bbu acts in parallel with Frizzled, perhaps by potentiating or stabilizing its negative effect on Notch, the alternative that Bbu acts in the Frizzled pathway upstream of Notch cannot be ruled out. Because Notch has been shown to participate in the specification of essentially all cell types of the eye disc, the reduced numbers of photoreceptors in Bbu null mutant clones may also be due to increased activities of the Notch pathway during different steps of eye development. Taken together, the combined data from overexpression and loss-of-function experiments make a strong case for a role of Bbu in downregulating Notch activity. However, by no means do they exclude the possibility that Bbu (and by extension, E(spl) m4, malpha, and Brd) acts in additional pathways that do not involve Notch signaling (Zaffran, 2000).

It is intriguing that Bbu was isolated as a result of its protein-protein interactions with the homeodomain protein Tinman. Although there is no formal proof that this interaction is relevant in vivo, several lines of evidence support its specificity, including the fact that three independent clones of Bbu cDNAs were isolated in the yeast two-hybrid screen and the observation that GST-Bbu fusion protein specifically associates with radiolabeled Tinman protein in in vitro binding assays. The observed co-expression of Bbu and Tinman in the early mesoderm would also favor the possibility of interactions of the two proteins in vivo. If this interaction were to occur, it would imply a nuclear function of Bbu, presumably as a cofactor of Tin in the mesoderm and additional transcription factors in other tissues. In this hypothesis, Bbu would interfere with transcriptional outputs of the Notch signaling cascade that require tissue-specific transcription factors such as Tinman. For example, it is possible that activated Notch/Su(H) complexes need to bind to enhancer sequences of certain target genes together with Tinman to activate their expression in the mesoderm, and that the binding of Bbu to Tin interferes with this cooperative activity. Indeed, tin-dependent specification events in heart and somatic muscle development also involve Notch signaling. While the predominantly cytoplasmic localization of Bbu protein seems to argue against an interaction with Tin in vivo, it is still possible that the nuclei contain low levels of Bbu protein that cannot be detected above background levels with antibody stainings (Zaffran, 2000).


REFERENCES

Search PubMed for articles about Drosophila Enhancer of split m4, Bearded family member

Apidianakis, Y., et al. (1999). Overexpression of the m4 and malpha genes of the E(spl)-Complex antagonizes Notch mediated lateral inhibition. Mech. Dev. 86(1-2): 39-50.

Bailey, A. M., Posakony, J. W. (1995). Suppressor of Hairless directly acti- vates transcription of Enchancer of split Complex genes in response to Notch receptor activity. Genes Dev. 9: 2609-2622.

Godt, D., et al. (1991). The distribution of transcripts of neurogenic genes in neurogenic mutants of Drosophila melanogaster. J. Neurogenet. 7(4): 241-52.

Klambt, C., et al. (1989). Closely related transcripts encoded by the neurogenic gene complex enhancer of split of Drosophila melanogaster. EMBO J. 8(1): 203-10. 89231619

Knust, E., Tietze, K. and Campos-Ortega, J. A. (1987). Molecular analysis of the neurogenic locus Enchancer of split of Drosophila melanogaster. EMBO J. 6: 4113-4123

Lai, E. C. and Posakony, J. W. (1998a). The Bearded box, a novel 3' UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression. Development 124(23):4847-56.

Lai, E. C., Burks, C. and Posakony, J. W. (1998b). The K box, a conserved 3' UTR sequence motif, negatively regulates accumulation of enhancer of split complex transcripts. Development 125(20): 4077-88.

Lai, E. C., et al. (2000). Antagonism of Notch signaling activity by members of a novel protein family encoded by the Bearded and Enhancer of split gene complexes. Development 127: 291-306.

Lai, E. C. (2002). Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation. Nat. Genet. 30(4): 363-4. 11896390

Lai, E. C., Tam, B. and Rubin, G. M. (2005). Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs. Genes Dev 19(9): 1067-1080. PubMed ID: 15833912

Leviten et al., (1997). The Drosophila gene Bearded encodes a novel small protein and shares 3' UTR sequence motifs with multiple Enhancer of split complex genes. Development 124(20): 4039-4051.

Nagel, A. C., et al. (2000). Neural hyperplasia induced by RNA interference with m4/malpha gene activity. Mech. Dev. 98: 19-28.

Nellesen, D. T., Lai, E. C. and Posakony, J. W. (1999). Discrete enhancer elements mediate selective responsiveness of enhancer of split complex genes to common transcriptional activators. Dev Biol 213(1): 33-53.

Schrons, H., Knust, E. and Campos-Ortega, J. A. (1992). The Enhancer of split complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells. Genetics 132(2): 481-503.

Singson, A., Leviten, M. W., Bang, A. G., Hua, X. H. and Posakony, J. W. (1994). Direct downstream targets of proneural activators in the imaginal disc include genes involved in lateral inhibitory signaling. Genes Dev. 8: 2058-2071

Wurmbach, E., Wech, I. and Preiss. A. (1999). The Enhancer of split complex of Drosophila melanogaster harbors three classes of Notch responsive genes. Mech. Dev. 80(2): 171-80.

Zaffran, S. and Frasch, M. (2000). Barbu: an E(spl) m4/malpha-related gene that antagonizes Notch signaling and is required for the establishment of ommatidial polarity. Development 127: 1115-1130.


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date revised: 5 January 2001

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