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).