Promoter Structure

DNA sequence reveals a consensus TATA box (TATTTAA) 30 bp upstream of the first base of the cDNAs (Singson, 1994).

Several genes are direct downstream targets of achaete-scute activation, as judged by the following criteria: the genes are expressed in the proneural clusters (PNCs) of the wing imaginal disc in an ac-sc-dependent manner; (2) the proximal promoter regions of all of these genes contain one or two high-affinity ac-sc binding sites, which define the novel consensus GCAGGTG(T/G)NNNYY, and (3) where tested, these binding sites are required in vivo for PNC expression of promoter-reporter fusion genes. Interestingly, these ac-sc target genes, including , Enhancer of split m7, Enhancer of split m8, and scabrous, are all known or believed to function in the selection of a single SOP from each PNC, a process mediated by inhibitory cell-cell interactions. Thus, one of the earliest steps in adult peripheral neurogenesis is the direct activation by proneural proteins of genes involved in restricting the expression of the SOP cell fate (Singson, 1994). Brd bristle phenotypes are dependent on ac/sc function (Leviten, 1996), and Brd transcript accumulation within wing disc proneural clusters likewise requires ac/sc activity. Brd promoter, containing the novel sequence described above. is directly activated in proneural clusters by bHLH protein complexes that include ac and sc (Singson, 1994). However, in contrast to ac and sc, there is generally no clear elevation of Brd transcript in presumptive SOPs (Leviten, 1997).

Targets of Activity

The 3' untranslated regions (3' UTRs) of Bearded, hairy, and many genes of the E(spl)-C contain a novel class of sequence motif, the GY box (GYB, GUCUUCC); extra macrochaetae contains the variant sequence GUUUUCC. The 3' UTRs of three proneural genes include a second type of sequence element, the proneural box (PB, AAUGGAAGACAAU). The full 13 nt PB is found once each in ac, l'sc, and ato, along with a second, variant version in both l'sc and ato. The presence of these motifs in such distantly related paralogs as hairy and certain bHLH genes of the E(spl)-C (for the GYB), and ato and two genes of the AS-C (for the PB), indicates that both classes of sequence element are subject to strong selection. Furthermore, both the PB and the GYB are conserved in the orthologs of ac and E(spl)m4 from the distantly related Drosophilids D. virilis and D. hydei, respectively, though these 3' UTRs are otherwise quite divergent from their D. melanogaster counterparts. These findings strongly suggest functional roles for both of these sequence elements (Lai, 1998).

Intriguingly, the central 7 nt of the PB and the GYB are exactly complementary, and are often located within extensive regions of RNA:RNA duplex predicted to form between PB- and GYB-containing 3' UTRs. Indeed, using in vitro assays, RNA duplex formation has been observed between the ato/Brd and ato/m4 3' UTR pairs that is PB- and GYB-dependent. It is noteworthy that the predicted duplex interactions involving the GYB of Brd are significantly stronger than those involving the GYBs of the other transcripts. For example, Brd and ato are perfectly complementary over 18 contiguous nucleotides. This difference in the degree of PB:GYB-associated complementarity is likely to have functional consequences (Lai, 1998).

In C. elegans, small antisense RNAs encoded by lin-4 mediate translational repression of lin-14 and lin-28 transcripts by binding to complementary sequences in their 3' UTRs. In Drosophila, PB- and GYB-bearing transcripts may likewise participate in a regulatory mechanism mediated by RNA:RNA duplexes, but with the feature that both partners are mRNAs that also direct the synthesis of functionally interacting proteins. The opportunity to form such duplexes clearly exists, since transcripts from proneural genes and their regulators very frequently accumulate in coincident or overlapping patterns. Moreover, while 7 nt is the minimum length of complementarity between any PB and any GYB, the longest possible uninterrupted duplex between a given GYB-bearing transcript and a given proneural partner is almost always considerably longer (8-12 nt). It is worth noting that in a lin-4/lin-14 duplex that has been shown to be sufficient for proper regulation in vivo, the longest region of uninterrupted complementarity is only 7 nt (Lai, 1998 and references therein).

The formation of the postulated RNA duplexes may serve to regulate proneural gene function, consistent with the known roles of hairy, emc, and the bHLH genes of the E(spl)-C. This might explain occasional C-to-U transitions in the GYB sequence (in emc and D. hydei m4); these variants retain complementarity with the PB due to G:U base-pairing. It is equally plausible that GYB-containing transcripts are regulated by duplex formation. A third very interesting possibility is that RNA:RNA duplexes formed between PB- and GYB-containing transcripts function to initiate a downstream regulatory activity affecting as-yet-unknown targets. Ample precedent exists establishing the trans-regulatory potency of double-stranded RNA. In any case, the apparent capacity of transcripts from the proneural genes and their regulators to form duplexes in their 3' UTRs suggests further complexity in the already complex regulatory interactions that control Drosophila neurogenesis (Lai, 1998).

Posttranscriptional regulation

Wild-type Brd 3' UTR confers negative regulatory activity on heterologous transcripts in vivo; 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; 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, it is found that 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, 1997).

The wild-type Brd 3' UTR includes three copies of a 9-nucleotide sequence (CAGCTTTAA) that is referred to as the 'Brd box'. The 3' UTRs of Brd and of the m4 transcription unit of the Enhancer of split gene complex [E(spl)-C] exhibit an unusually high degree of sequence identity that includes not only Brd box sequences but also a second motif referred to as the 'GY box' (GTCTTCC). Both the Brd box and the GY box are also present in the 3' UTRs of several basic helix-loop-helix repressor-encoding genes of the E(spl)-C, often in multiple copies, suggesting that a novel mode of post-transcriptional regulation applies to Brd and many E(spl)-C genes. The fact that the more abundant Brd mutant mRNA lacks the GY box and that two of the Brd boxes are present in wild-type Brd mRNA suggests that either or both of these elements may confer instability on transcripts that contain them (Leviten, 1997).

RNA silencing phenomena, either the regulation of mRNA translation or regulation of mRNA degradation, intersect at the ribonuclease Dicer. In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. siRNAs and miRNAs then direct a RNA-induced silencing complex (RISC) to cleave mRNA or block its translation (RNAi). Mutations have been characterized in the Drosophila dicer-1 and dicer-2 genes. Mutation in dicer-1 blocks processing of micro RNA precursors, whereas dicer-2 mutants are defective for processing siRNA precursors. It has been recently found that Drosophila Dicer-1 and Dicer-2 are also components of siRNA-dependent RISC (siRISC). Dicer-1 and Dicer-2 are required for siRNA-directed mRNA cleavage, though the RNase III activity of Dicer-2 is not required. Dicer-1 and Dicer-2 facilitate distinct steps in the assembly of siRISC. However, Dicer-1 (but not Dicer-2) is essential for miRISC-directed translation repression. Thus, siRISCs and miRISCs are different with respect to Dicers in Drosophila (Lee, 2004).

Although Dcr-1 and Dcr-2 preferentially produce different types of small RNAs, both are required for efficient siRNA-dependent mRNA degradation. Does this dual requirement extend to the miRNA pathway as well? To test this possibility, a genetic assay for miRNA-dependent gene silencing was used in dcr-1 and dcr-2 mutants. Several classes of motifs are present in the 3'UTR regions of the E(spl) and Bearded genes. The 3'UTR motifs are complementary to a variety of miRNAs, and they mediate posttranscriptional repression of gene expression. A series of reporter transgenes was constructed that mimics this posttranscriptional repression. The reporter genes contain a constitutive promoter from armadillo, lacZ coding sequence, and the 3'UTR from the Bearded or E(spl)m8 gene. When the reporter contains a wild-type Bearded 3'UTR, its expression in the developing eye disc is very weak. It is somewhat more strongly expressed in the eye disc posterior to the morphogenetic furrow and is equally weak in the anterior eye disc and antennal disc. When the reporter contains a Bearded 3'UTR with its three B motifs mutated, expression is ubiquitously strong in the eye and antennal discs, confirming that the B motifs mediate a silencing effect on gene expression (Lee, 2004).

Expression of a wild-type reporter gene was examined in clones of mutant dcr-2 cells that were generated in the developing eye disc. Clones expressed the reporter at levels indistinguishable from wild-type tissue, indicating that Dcr-2 is not required for this gene silencing mechanism. In contrast, expression of a wild-type reporter gene in clones of mutant dcr-1 cells was much stronger than in wild-type tissue. The derepressive effect of the dcr-1 mutation requires intact B motifs in the Bearded 3'UTR, since mutant clones did not affect expression of a reporter gene with mutated B motifs. These results argue that dcr-1 but not dcr-2 is necessary for posttranscriptional gene silencing that is mediated by a miRNA mechanism. This conclusion is also validated by other mutant phenotypes associated with each gene. Loss of dcr-1 has profound effects on Drosophila development within both somatic- and germ-lineages, whereas loss of dcr-2 appears to have little or no effect on development (Lee, 2004).

Pervasive regulation of Drosophila Notch target genes by GY-box-, Brd-box-, and K-box-class microRNAs

Although hundreds of distinct animal microRNAs (miRNAs) are known, the specific biological functions of only a handful are understood at present. Three different families of Drosophila miRNAs directly regulate two large families of Notch target genes, including basic helix-loop-helix (bHLH) repressor and Bearded family genes. These miRNAs regulate Notch target gene activity via GY-box (GUCUUCC), Brd-box (AGCUUUA), and K-box (cUGUGAUa) motifs. These are conserved sites in target 3'-untranslated regions (3'-UTRs) that are complementary to the 5'-ends of miRNAs, or 'seed' regions. Collectively, these motifs represent >40 miRNA-binding sites in Notch target genes, and all three classes of motif are shown to be necessary and sufficient for miRNA-mediated regulation in vivo. Importantly, many of the validated miRNA-binding sites have limited pairing to miRNAs outside of the "box:seed" region. Consistent with this, it was found that seed-related miRNAs that are otherwise quite divergent can regulate the same target sequences. Finally, it is demonstrated that ectopic expression of several Notch-regulating miRNAs induces mutant phenotypes that are characteristic of Notch pathway loss of function, including loss of wing margin, thickened wing veins, increased bristle density, and tufted bristles. Collectively, these data establish insights into miRNA target recognition and demonstrate that the Notch signaling pathway is a major target of miRNA-mediated regulation in Drosophila (Lai, 2005).

The E(spl)-C and Brd-C of Drosophila melanogaster (Dm) contain two large families of direct Notch target genes, including seven bHLH repressor-encoding genes and 10 Bearded family genes. With the exception of E(spl)mbeta and Ocho, all of these genes contain GY-box (GUCUUCC), Brd-box (AGCUUUA), and/or K-box (UGUGAU) motifs in their 3'-UTRs, which are propose to be miRNA-binding sites. Nine of these genes contain three or more box sites, a density that is especially remarkable when one considers how short their 3'-UTRs are (often <350 nt in length). The conservation of these sites were systematically assessed in their orthologs from Drosophila pseudoobscura (Dp) and Drosophila virilis (Dv), species that are ~30 million and 60 million years diverged from Dm, respectively. 33/51 Brd-boxes, GY-boxes, and K-boxes have been perfectly conserved and reside in syntenic locations among all three species; 11 additional sites are identical in two of the three species. This indicates that all three motifs are under strong selective constraint (Lai, 2005).

Closer examination of nucleotide divergence surrounding these boxes has revealed some unexpected features that are germane to the proposition that these boxes represent miRNA-binding sites. These features are best illustrated by comparing rapidly evolving genes. Notably, Bearded is an unusually rapidly evolving protein, with only 15 residues preserved between Dm and Dv orthologs (out of 81 and 66 amino acids, respectively), and Dv Bearded has a significantly different arrangement of these 3'-UTR motifs. The 3'-UTR of Dv E(spl)m5 is also quite different from its counterparts in Dm/Dp. Alignment of Dm/Dp orthologs of Bearded and E(spl)m5 reveals that sequences upstream of most GY-boxes are well conserved; these regions include most sequences presumed to pair with miR-7. Similar patterns are seen for many other GY-boxes in other Notch target genes. However, the sequence upstream of many Brd- and K-boxes is strongly diverged, so that only 'box'-pairing is often preserved. In fact, many Brd- and K-boxes generally lack extensive pairing to miRNAs outside of the 'box' sequence. These factors likely preclude their identification by various published computational algorithms for miRNA-binding sites. Indeed, Brd- and K-boxes in Notch target genes have been deemed unlikely to represent miRNA-binding sites. In contrast, rapid divergence of the upstream portion of miRNA-binding sites is consistent with the idea that pairing between the miRNA "seed" (positions ~2-8) and the 3'-UTR 'box' (approximately the last one-third of the miRNA-binding site) is most critical for miRNA-mediated regulation (Lai, 2005).

It is also noted that precise spacing of several motif occurrences that are closely paired is also conserved, even though orthologous 3'-UTRs otherwise display significant insertions and deletions. In these cases, one would presume that simultaneous binding of miRNAs to their respective sites would not be possible unless the 3'-end of the downstream miRNA was unpaired, a configuration that unexpectedly proved functional in vitro. Finally, there are a few nonconserved boxes in these 3'-UTRs (7/51 total sites). In several cases, the nonconserved site is highly related to a neighboring conserved site [i.e., the first and second GY-boxes of Dp E(spl)m4 are equally similar to the first GY-box in Dm E(spl)m4; the third and fourth Brd-boxes in Dp E(spl)m5 are highly related to the third Brd-box in Dm E(spl)m5], implying that these nonconserved sites may be functional, newly evolved miRNA-binding sites (Lai, 2005).

GY-box-, Brd-box-, and K-box-class miRNAs are highly conserved among diverse insects, and many are, indeed, identical. Therefore Brd-boxes, GY-boxes, and K-boxes were sought in the predicted 3'-UTRs of E(spl)bHLH and Brd genes from mosquitoes, bees, and moths; these species cover ~350 million years of divergence from Drosophila. Impressively, homologs of both E(spl)bHLH and Brd genes in these highly diverged species all contain multiple copies and multiple classes of 'box' motifs in their 3'-UTRs. This strongly suggests that regulation by all three families of miRNAs is an ancient feature of Notch target gene regulation in insects (Lai, 2005).

To directly test the capacity of miRNAs to regulate the 3'-UTRs of these Notch target genes, an in vivo assay was used. The target in this assay is a ubiquitously expressed reporter (tub>GFP or arm>lacZ) fused to an endogenous 3'-UTR (a 3'-UTR sensor). The reporter transgene is introduced into a genetic background in which a UAS-DsRed-miRNA transgene is activated with dpp-Gal4 or ptc-Gal4. This results in ectopic miRNA production in a stripe of red-fluorescing cells at the anterior–posterior boundary of imaginal discs. Inhibition of the green reporter within the red miRNA-misexpressing domain reflects direct miRNA-mediated negative regulation. Focus was placed on the central wing pouch region of the wing imaginal disc (Lai, 2005).

The ability of sensor transgenes for most Bearded family genes [Bob, Bearded, Tom, Ocho, E(spl)malpha, and E(spl)m4] and most E(spl)bHLH repressor genes [E(spl)mgamma, E(spl)mdelta, E(spl)m3, E(spl)m5, and E(spl)m8] to be regulated by ectopic GY-box-, Brd-box-, and K-box-class miRNAs was extensively analyzed. Sensor expression is influenced by the level to which it is negatively regulated by endogenous factors, including miRNAs. In this assay, the disc sensor must be expressed at sufficient levels before one can observe its knock-down by ectopic miRNAs. 3'-UTR sensor constructs for different Notch target genes accumulate to different levels in vivo, consistent with variable amounts of endogenous miRNA-mediated regulation. Nevertheless, it was possible to reliably detect expression of all sensors excepting E(spl)m8. As detailed in the following three sections, these sensors were used to unequivocally demonstrate GY-boxes, Brd-boxes, and K-boxes to be sites of miRNA-mediated negative regulation by corresponding families of complementary miRNAs in vivo (Lai, 2005).

miR-7 is the only known Drosophila miRNA whose 5'-end is complementary to the GY-box (GUCUUCC). miR-7 has been shown to regulate three GY-box targets, including two members of the E(spl)-C, E(spl)m3 and E(spl)m4. While these two genes scored well in a genome-wide prediction of miR-7 targets, many other members of the Brd-C and E(spl)-C also contain between one and three GY-boxes in their 3'-UTRs [Bob, Bearded, Tom, E(spl)mgamma, E(spl)m5]. Of these, only Tom was computationally identified as a compelling candidate for miR-7 (Lai, 2005).

The specificity of the disc sensor assay was assayed by showing that neither an empty tub-GFP sensor nor an Ocho sensor were affected by miR-7. The previous experiments done with E(spl)m3 and E(spl)m4 were repeated and it was observed that both were, indeed, inhibited by ectopic miR-7. This assay was used to demonstrate that miR-7 negatively regulates all seven GY-box-containing members of the Brd-C and E(spl)-C, including those with single sites [E(spl)m3, E(spl)mgamma, and Bearded], those with two sites [E(spl)m4, Tom, Bob], and those with three sites [E(spl)m5]. These data convincingly support the hypothesis that GY-boxes are general signatures of miR-7-binding sites in Notch target genes, irrespective of the overall amount of pairing between miR-7 and sequences outside of the GY-box. In order to more definitively demonstrate that miR-7-mediated regulation occurs through identified GY-boxes, mutant sensors bearing point mutations in the GY-boxes were tested. A Bearded sensor carrying five point mutations in its single GY-box no longer responded to miR-7. In a more stringent test, an E(spl)m5 sensor carrying 2-nt mutations in each of its three GY-boxes was generated. These targeted changes also abolished the ability of miR-7 to negatively regulate E(spl)m5. Therefore, ~7 continuous base pairs between the 'box' motif and its cognate miRNA seed are critical for in vivo target regulation. It is also noted that when mutant 3'-UTRs are tested, a mild increase in reporter activity in miRNA-misexpressing cells was sometimes observed, the reason for which has not been determined (Lai, 2005).

Previous work has suggested synergism between miRNA-binding sites on the same transcript. Multiple GY-box 3'-UTRs were generally subject to greater regulation than single-site 3'-UTRs, even though the amount of miR-7 pairing to individual GY-boxes in multiple-site 3'-UTRs is often less than its pairing with single GY-box 3'-UTRs. Indeed, negative regulation of E(spl)m4, Tom, Bob, and E(spl)m5 by miR-7 was qualitatively indistinguishable from an artificial sensor containing two perfectly miR-7-complementary sites, even though many sites in these genes display relaxed pairing with miR-7 outside of GY-boxes. This suggests that as little as 7–8 nt of complementarity may suffice for miRNA target recognition, especially where multiple sites are present. However, since all three single GY-box-containing 3'-UTRs were also regulated by miR-7, synergism is not required for biologically significant regulation by miRNAs (Lai, 2005).

There are two Drosophila miRNAs, miR-4 and miR-79, whose 5'-ends are complementary to the Brd-box (AGCUUUA). Both miRNAs are resident in miRNA clusters, and miR-4 resides in particularly dense clusters containing several unrelated miRNAs. Use was made of a UAS-DsRed-miR-286, miR-4, miR-5 transgene that is referred to as "UAS-miR-4" and a UAS-DsRed-miR-79 transgene. miR-4 and miR-79 have only limited similarity outside of their Brd-box seed, and there is little indication from pairwise alignments that these miRNAs are specifically "tuned" to different Brd-box sites in Notch target genes. In fact, all of these Brd-boxes lack the extended complementarity to miRNAs that is typical of miR-7:GY-box pairs, and no Notch target genes were previously predicted computationally as targets of miR-4 or miR-79 (Lai, 2005).

Seven Brd-box-containing Notch target genes were validated as being regulated by Brd-box-family miRNAs, including those with single sites [Tom, E(spl)mdelta, E(spl)mgamma] and those with multiple sites [Bearded, E(spl)malpha, E(spl)m4, and E(spl)m5]. Curiously, the negative regulatory effects of miR-4 on E(spl)mgamma, E(spl)malpha, E(spl)m4, and E(spl)m5 were greater than those of miR-79 on these same 3'-UTRs, even though miR-4 is no more complementary to these sites than is miR-79. Nevertheless, the common ability of miR-4 and miR-79 to down-regulate individual sensors indicates that cross-regulation of individual sites by multiple members of a given miRNA family may occur. Notably, both miRNAs are expressed at high levels during embryonic development (Lai, 2005).

The specificity of miR-4 and miR-79 was tested using two mutant Bearded sensors, one bearing several point mutations in each of its three Brd-boxes and another containing mutations in the Brd-boxes and the GY-box. In both cases, the mutant transgenes accumulate to higher levels, consistent with relief from negative regulation by endogenous Brd-box-class miRNAs in the wing disc. In addition, they are no longer responsive to ectopic Brd-box-class miRNAs, indicating that the observed regulation occurs directly via Brd-boxes. As well, this experiment demonstrates that regulation by the miR-4 transgene is not attributable to miR-286 and miR-5 carried on this construct. Nevertheless, this miRNA construct efficiently down-regulates a miR-5 sensor containing two miR-5 sites, indicating that the other miRNAs carried on this construct are functional. As a final test of the specificity of this assay, it was observed that this three-miRNA construct fails to inhibit the expression of an empty tub-GFP sensor (Lai, 2005).

Having demonstrated that Brd-boxes are bona fide miRNA-binding sites, it was asked whether regulation of the Bearded 3'-UTR by miR-7 requires the presence of Brd-boxes. This might be the case, for example, if negative regulation of a given 3'-UTR required synergism between different types of miRNA-binding sites. A Bearded 3'-UTR carrying mutations in each of the three Brd-boxes was observed to be still strongly inhibited by miR-7, indicating that individual types of miRNA-binding sites suffice for regulation in this assay (Lai, 2005).

The largest family of Drosophila miRNAs includes those whose 5'-ends are complementary to the K-box (cUGUGAUa, where the lowercase nucleotides represent positions of strong bias). The K-box is also the most pervasive motif within these Notch target genes; it is present in almost every member of the Brd-C and E(spl)-C [excepting E(spl)mbeta and Ocho, which lack any box motifs]. The maximum overall site complementarity of any given K-box site to any K-box family miRNAs is generally modest, and less than that seen with other demonstrated targets of the K-box family miRNA miR-2, namely, the proapoptotic genes grim, reaper, and sickle. In fact, the sole Notch target gene that was predicted informatically as a target of a K-box family miRNA in any study was E(spl)m8: miR-11 , and this pair ranked only 46th (Lai, 2005).

The ability was tested of two quite distinct K-box family miRNAs, those of the miR-2 cluster (miR-2a-1, miR-2a-2, and miR-2b-2) and miR-11, to regulate K-box-containing 3'-UTRs. Given the abundance of K-box complementary miRNAs (as a class, they are among the more frequently cloned fly miRNAs), the occupancy of K-box sites by endogenous K-box-class miRNAs may be near-saturating in some cases. In fact, negative regulation of E(spl)m8, whose K-boxes mediate 10-fold negative regulation and nearly eliminate expression of this sensor, could not be convincingly demonstrated. In spite of this, positive evidence was obtained that four other K-box-containing 3'-UTRs, E(spl)m4, Bob, E(spl)malpha, and E(spl)mdelta, are directly regulated by K-box-family miRNAs, although the amount of regulation observed was weaker than that seen with GY-box- or Brd-box-class miRNAs. As was the case with the two Brd-box-class miRNAs, both miR-2 and miR-11 are capable of regulating some of the same K-box-containing targets. This constitutes further evidence for the possibility of cross-regulation of miRNA-binding sites, even where the miRNAs in question display very little similarity outside of their seeds (Lai, 2005).

In performing pairwise tests of these miRNAs with Notch target gene sensors, two instances were observed of miRNA-mediated regulation of sensors lacking canonical boxes. (1) It was observed that the E(spl)mdelta sensor was inhibited by miR-7. Although E(spl)mdelta lacks a canonical GY-box, it does contain a GY-box-like site that would have a single G:U base pair with the miR-7 seed. The nucleotides that are 5' and 3' to the box are also paired with miR-7, and there is a significant region of pairing to the 3'-end of the miRNA. These factors may allow this site to be recognized by miR-7. The 9-mer AGUUUUCCA is found in both Dp and Dv orthologs of E(spl)mdelta, indicating that this site is under selection and therefore is likely important for regulation of E(spl)mdelta. (2) It was observed that the Bob sensor was negatively regulated by both Brd-box-class miRNAs, miR-4 and miR-79. Although Bob lacks a canonical Brd-box, it does contain two matches to positions 2-7 of the Brd-box, which would pair to positions 2-7 of the miR-4/79. In this regard, this type of site is reminiscent of the 6-mer K-box, which pairs to positions 2-7 of K-box miRNAs. One of these Brd-box-like sites is conserved in Dp, and the syntenic site in Dv is, in fact, a canonical Brd-box, further indicating a functional relationship between Bob and miRNAs of the Brd-box family (Lai, 2005).

The apparent functionality of these noncanonical sites led to a search for other such sites in Notch target 3'-UTRs. Although one might expect to find many-fold more copies of degenerate sites relative to canonical sites, instead only a few additional examples of relaxed GY-box-like or Brd-box-like sites were found. For comparison, there are 28 canonical sites of these classes in Notch target 3'-UTRs (16 Brd-boxes and 12 GY-boxes), but only three additional examples of a 7-mer box-like site with a G:U base-pair to a miRNA seed [all are GY-box-like sites in E(spl)mdelta, E(spl)m3, and E(spl)m7]. In addition, there are only five additional examples of sites that match only positions 2-7 of the GY-box or the Brd-box [all of which are Brd-box-like sites: the two in Bob, one in E(spl)m7, one in E(spl)malpha, and one in E(spl)mdelta]. These considerations strongly suggest that the much more restricted, canonical sites are actively selected for function in these Notch target 3'-UTRs, a conclusion that is bolstered by the patterns of evolutionary conservation of these sites (Lai, 2005).

These experiments presented thus far demonstrate that target gene 3'-UTRs harboring sequence elements with Watson-Crick complementarity to the 5'-ends of miRNAs are, indeed, regulated by these miRNAs in vivo, and that such sites are necessary for miRNA-mediated regulation. Are these sites sufficient for regulation by complementary miRNAs? Although a variety of studies of model sites in tissue culture assays indicate site sufficiency, tests in animals suggest that miRNA site context can be less forgiving in vivo. For example, certain reporters containing multimers of six lin-4 or three let-7 sites are not appropriately regulated by lin-4 or let-7 in nematodes. In addition, mutation of sequences outside of the let-7-binding sites in lin-41 abolishes regulation by let-7 in vivo. Therefore, it was of interest to test the functionality of GY-boxes, Brd-boxes, and K-boxes when abstracted from endogenous 3'-UTR context (Lai, 2005).

To do so, a tandem of isolated GY-box, Brd-box, and K-box elements were cloned from Bob, Bearded, and E(spl)m8, respectively, into tub-GFP transgenes. Also mutant versions were cloned containing single changes in the Brd-box sites or dual changes in the GY-boxes. The ability of these 'box' sensors to respond to exogenously expressed miRNAs was tested. It was found that wild-type GY-box, Brd-box, and K-box sensors are all negatively regulated by corresponding miRNAs. These data directly demonstrate that all three types of box sites are sufficient for miRNA-mediated negative regulation. In contrast, mutant box sensors are nonfunctional in this assay. Since the mutant box sensors contain only one or two changes in each site, these data provide strong in vivo support for the idea that Watson-Crick pairing to the 5'-end of the miRNA (the "seed") is the key essential feature of miRNA target recognition. As a further test of this idea, the ability of the three different K-box miRNAs, miR-6, miR-2, and miR-11, to down-regulate a miR-6 sensor was tested. All three inhibited miR-6 sensor expression, consistent with the ability of seed-pairing to mediate regulation by miRNAs (Lai, 2005).

With these UAS-miRNA transgenic lines in hand, the consequences of ectopically expressing miRNAs on Drosophila development were tested. It should be noted that Notch target-regulating miRNAs were fully expected to regulate other functionally unrelated targets in vivo. For example, it has been established that K-box-family miRNAs also negatively regulate the proapoptotic genes reaper, sickle, and grim via K-boxes in their 3'-UTRs, while Brd-box-family miRNAs target the mesodermal determinant bagpipe via a Brd-box in its 3'-UTR. Therefore, even if ectopic miRNAs are able to affect normal development, they would not necessarily be expected to affect Notch signaling exclusively. Nevertheless, it has been previously reported that ectopic miR-7 induces loss of molecular markers of wing margin development, resulting in wing notching. This indicates that phenotypic characterization of miRNA misexpression can be informative (Lai, 2005).

Using an independently derived UAS-miR-7 construct lacking DsRed, it was verified that dpp-Gal4>miR-7 wings display notching and loss of Cut expression at the developing wing margin of wing imaginal discs; the size of the L3-L4 intervein domain was also reduced. It was next observed that ectopic K-box miRNAs of the miR-2a-1, miR-2a-2, miR-2b-2 cluster or miR-6-1, miR-6-2, miR-6-3 cluster had similar effects on wing margin development, although two UAS-transgenes were necessary to produce this effect. Also loss of anterior crossvein and occasional L3 vein breaks was observed, although these are not indicative of loss of N signaling. More generalized expression of miR-7 using bx-Gal4 induced strong thickening of wing veins, which is indicative of compromised Notch signaling during lateral inhibition of wing veins. Expression of K-box miRNAs using bx-Gal4 had severe effects on wing development, resulting in tiny, crumpled wings. It is suspected that this results from misregulation of non-Notch-pathway-related targets. The Brd-box miRNAs miR-4 and miR-79 and the K-box miRNA miR-11 did not affect wing margin development, even when these transgenes were present in two copies, indicating that this phenotype is not generally due to misexpression of miRNAs. However, miR-79 induced strong wing curling at high levels, potentially due to misregulation of non-Notch-pathway-related targets (Lai, 2005).

Next, focus was placed on development of the adult peripheral nervous system, as exemplified by the bristle sensory organs that decorate the body surface. A classic role for Notch signaling is to restrict the number of sensory organ precursors. It was found that misexpression of miR-6 using bx-Gal4 results in a strong increase in microchaete bristle density and clustered dorsocentral macrochaetes, phenotypes that are consistent with loss of Notch signaling during lateral inhibition of sensory organ precursors. Ectopic miR-2 had a similar, but milder, effect and mostly induced clustered dorsocentral and scutellar macrochaetes. Therefore, divergent members of the K-box miRNA family have similar effects on sensory organ development, consistent with data indicating that seed-related miRNAs can regulate overlapping sets of target genes. Ectopic miR-7 also induces macrochaete tufting, which correlates with the differentiation of supernumerary sensory organ precursors in wing imaginal discs. Finally, occasional duplication of bristles was observed upon misexpression of the Brd-box miRNA mir-79, although this construct also induced occasional bristle loss. Ectopic expression of miRNAs does not in itself induce bristle defects per se, since misexpression of miR-4 or miR-11 does not interfere with bristle development (Lai, 2005).

Overall, the ability of different classes of Notch-regulating miRNAs to specifically induce phenotypes that are characteristic of Notch pathway loss of function in multiple developmental settings is a strong indication that Notch pathway targets validated in this study are key endogenous targets of these miRNAs (Lai, 2005).

It appears, therefore, that cells go through a significant amount of trouble to actively inhibit Notch signaling. Core components of the Notch pathway are subject to significant negative regulation at every step in their life cycle, including at the transcriptional, post-transcriptional, and post-translational levels. For example, in the absence of activated nuclear Notch, CSL proteins are transcriptional repressors that actively repress Notch target gene activity. In addition, multiple dedicated ubiquitin ligases promote degradation of Notch pathway components, including the receptor Notch itself. To this list, may be added transcripts of most direct Notch target genes in Drosophila that are negatively regulated by multiple families of miRNAs (Lai, 2005).

The evidence provided in this study to support this conclusion is that (1) three different classes of miRNA-binding sites (GY-boxes, Brd-boxes, and K-boxes) are pervasive among two major classes of Notch target genes; (2) all three classes of motif are selectively constrained in 3'-UTRs during evolution; (3) transcripts bearing these box sites are negatively regulated by complementary miRNAs in vivo; (4) all three classes of sites are both necessary and sufficient for miRNA-mediated regulation in vivo; and (5) ectopic expression of Notch target-regulating miRNAs phenocopies Notch pathway loss of function during multiple developmental settings. Perhaps most importantly, it has been shown that genomic transgenes specifically mutated for miRNA-binding sites are sufficiently hyperactive so as to perturb normal development of the peripheral nervous system. This places these Drosophila Notch target genes in a relatively select group of miRNA targets for which miRNA-mediated regulation is phenotypically essential for normal development (Lai, 2005).

While most of the previously characterized in vivo targets of miRNAs are of the 'extensive pairing' variety, many of the validated targets in this study display much more limited 'box:seed'-pairing to miRNAs. In fact, within the context of the set of Notch target gene 3'-UTRs, the presence of conserved GY-boxes, Brd-boxes, and K-boxes allowed for highly effective prediction of miRNA:target relationships. This is the case even without first taking into account the extent of miRNA-pairing outside of box motifs. Rapid divergence of sequences upstream of box motifs, particularly those of the Brd-box and K-box classes, further indicates that extensive pairing is not selected for in these bona fide target sites. Consistent with this, multiple lines of evidence are presented that show that divergent seed-related miRNAs can regulate overlapping sets of target in vivo. Conversely, the importance of pairing between 3'-UTR boxes to miRNA seeds was demonstrated by endogenous 3'-UTR and box sufficiency tests, where even single-nucleotide disruption of seed-pairing abolishes regulation by miRNAs in vivo (Lai, 2005).

Identification and characterization of miRNA-binding sites in these Notch target 3'-UTRs mesh well with other recent bioinformatics and experimental studies that together help to define the 'look' of miRNA-binding sites. The concept of using conserved 'boxes' with Watson-Crick complementarity to miRNA seeds to identify miRNA targets is at the heart of the TargetScanS approach. A recent study has identify statistically significant signal not only for conserved 3'-UTR sites that match positions 2-8 of the miRNA (as is characteristic of the Brd-box and GY-box), but also for matches to positions 2-7 of the miRNA (as is characteristic of the K-box). In addition, a significant bias was identified for the nucleotide corresponding to position one of the miRNA to be an adenosine in predicted target sites. Interestingly, 27/42 (64%) of GY-boxes, Brd-boxes, and K-boxes in Dm Notch target genes also have an adenosine in this position, consistent with the notion that this feature can help to identify genuine target sites. These results are also consistent with directed tests of model sites using an imaginal disc sensor assay. Together with the recent observation that miRNAs can down-regulate large numbers of transcripts that contain box:seed matches in their 3'-UTRs, a current view emerges that conserved 3'-UTR boxes that are 6-7 nt in length and complementary to the 5'-ends of miRNAs need to be considered seriously as functional regulatory sites. While seed-pairings with G:U base pairs are evidently not generally selected for, evidence is shown that rare sites of this class are functional. This is consistent with other studies that demonstrate that G:U seed-pairing impairs, but does not necessarily abolish target site function (Lai, 2005).

Finally, the presence of multiple classes of miRNA-binding sites in most Notch target gene 3'-UTRs raises the possibility of combinatorial regulation. Although this has been widely suggested as a formal possibility, extensive evidence has been provided that 3'-UTRs can bear multiple classes of functional sites. Phylogenetic considerations indicate that 10 different Notch target genes are likely regulated by multiple classes of miRNAs, and direct experimental support of this was provided for six Notch target genes. Multiple Brd-box-, K-box-, and GY-box-class miRNAs are present at high levels in the Drosophila embryo, and the Brd-box miRNA miR-4 is co-transcribed with the K-box miRNAs miR-6-1, miR-2, miR-3, suggesting that combinatorial control of Notch target genes actually occurs during normal development. Future studies are aimed at examining how different miRNA-binding sites collectively contribute to overall regulation of an individual gene (Lai, 2005).

Of the few animal miRNAs whose in vivo functions and targets are well understood, most act as genetic switches that determine binary, on/off states of target gene activity. For example, lin-4 and let-7 are temporal switches that control progression through nematode larval stages by inhibiting their targets at designated times in development. lsy-6 and miR-273 are spatial switches whose extremely restricted cell-type-specific expression patterns control neuronal identity. In these cases, temporally or spatially restricted miRNA expression is central to their control of specific processes, and each of these miRNAs appears to have a small number of key targets (Lai, 2005).

A different rationale is proposed for Brd-box and K-box miRNAs during Drosophila development. Although endogenous territories of GY-box-mediated regulation are not known, negative regulation by Brd-boxes and K-boxes appears spatially and temporally ubiquitous. Thus, Notch target transcripts of the Brd family and E(spl)bHLH families are subject to modes of miRNA-mediated regulation that operate in all cells, even though the genes themselves display highly restricted patterns of spatial expression. This suggests that these miRNAs are not dedicated to regulating Notch signal transduction, but may 'tune' the expression of many target genes. Indeed, the K-box-family miRNAs miR-2, miR-6, and miR-11 also directly regulate K-box-containing proapoptotic genes, and the Brd-box-family miRNAs miR-4 and miR-79 regulate the mesodermal determinant bagpipe. One prediction is that even though mutation of Brd-boxes and K-boxes in individual Notch target genes results in specific defects in Notch-mediated cell fate decisions, mutation of Brd-box and K-box miRNAs would have more general developmental consequences. This is supported by the observation that many, but not all, of the phenotypes induced by ectopic expression of Notch-regulating miRNAs appear to be obviously related to repression of Notch pathway activity (Lai, 2005).

An important advance of this study is the in vivo validation of a large number of biologically relevant miRNA targets that are minimally paired to miRNAs outside of the 'box:seed' region. Since modestly complementary sites are both necessary and sufficient for miRNA-mediated regulation, it might be relatively easy for novel miRNA-binding sites to arise in 'tuning' targets. Indeed, a subset of box sites has apparently newly evolved during Drosophilid radiation. In the greater context of insect Notch target genes, it appears to have been important that they be negatively regulated by miRNAs, although the precise numbers and arrangement of different sites is variable. These features of tuning targets seem to allow for highly customized regulation of individual genes (Lai, 2005).

The experimental validation of many tuning targets may be challenging or impossible to obtain where quantitative regulation is subtle. Nevertheless, minor changes in gene activity, even of a fraction of a percent, could become highly significant when selecting the fitness of individuals at the population level. Deep evolutionary profiling of related species will therefore be key to revealing the full complement of biologically important miRNA-binding sites. The data suggest that multiple classes of miRNA-binding sites can be recognized with confidence as highly conserved 3'-UTR 'boxes' complementary to miRNA seeds, and this approach has been applied to the analysis of mammalian genomes. By mid-2005, 12 Drosophila genomes will be completed, which should enable high-confidence identification of miRNA-binding sites on the genome-wide scale -- even in cases in which only 7 nt of the target are paired to a miRNA (Lai, 2005).

Recent computational work pointed to regulation of vertebrate Notch and Delta by miR-34; however, no Notch target genes were similarly singled out in various bioinformatics efforts. miR-34 is conserved in flies; however, inspection of fly Notch or its ligands Delta and Serrate failed to reveal 'boxes' that might indicate similar regulation by miR-34. Brd-box-, GY-box-, and K-box-complementary miRNAs are likewise conserved between flies and vertebrates. Are any vertebrate Notch target genes predicted to be targeted by these miRNAs by virtue of 'boxes'? Although Brd proteins have thus far been found only in insects, E(spl)bHLH proteins are conserved in and are primary effectors of Notch signaling in all vertebrates. No enrichment for Brd-boxes, GY-boxes, and K-boxes is observed across the set of vertebrate E(spl)bHLH 3'-UTRs as a whole. However, members of a specific subset of E(spl)-related repressors, named the Hey genes, contain a preponderance of these boxes in their 3'-UTRs. This appears to be the case in a variety of mammals (human, mouse, and rat) and fish (fugu and zebrafish). Therefore, miRNA-mediated regulation may be a conserved feature of Notch target genes, a scenario that is under current experimental investigation (Lai, 2005).

Bearded family members inhibit Neuralized-mediated endocytosis and signaling activity of Delta in Drosophila

Endocytosis of Notch receptor ligands in signaling cells is essential for Notch receptor activation. In Drosophila, the E3 ubiquitin ligase Neuralized (Neur) promotes the endocytosis and signaling activity of the ligand Delta (Dl). This study identifies proteins of the Bearded (Brd) family as interactors of Neur. Tom, a prototypic Brd family member, inhibits Neur-dependent Notch signaling. Overexpression of Tom inhibits the endocytosis of Dl and interferes with the interaction of Dl with Neur. Deletion of the Brd gene complex results in ectopic endocytosis of Dl in dorsal cells of stage 5 embryos. This defect in Dl trafficking is associated with ectopic expression of the single-minded gene, a direct Notch target gene that specifies the mesectoderm. It is proposed that inhibition of Neur by Brd proteins is important for precise spatial regulation of Dl signaling (Bardin, 2006).

In order to identify regulators of Neur, a yeast two-hybrid screen was conducted using as bait the conserved central domain of Neur that comprises the two Neur homology repeats (NHRs). Eighty-four cDNAs were identified, of which 62 encoded members of the Brd gene family: Ocho (39 times [×]), Tom (12×), m4 (7×), Brd (2×), and Bob (2×). Interaction of Neur with m6, mα, and m2 was tested directly. The m6 and mα proteins, but not m2, interacted with Neur in this assay. Tom also interacts with full-length Neur. It is concluded that all Brd family members, with the exception of m2, interact with Neur (Bardin, 2006).

Most Brd proteins share four conserved motifs: (1) a lysine-rich N-terminal region predicted to form an amphipathic α helix; (2) a short NxxNExLE motif, found in all proteins except m2; and (3 and 4) two C-terminal motifs found in only a subset of the Brd family members. Motifs 2 and 3 are the most-conserved motifs among insect Brd homologs. Because Brd and Bob lack motifs 3 and 4, these motifs cannot be strictly required for interaction with Neur. Additionally, clones encoding N-terminally truncated Tom proteins (amino acids 75-58, 58-158, 54-158, and 29-158 of Tom) were recovered in the screen, indicating that the N-terminal region predicted to form an amphipathic helix (amino acids 26-43 of Tom) is not necessary for interaction with Neur. Thus, motif 2 is the only conserved motif present in all the clones that interact with Neur. It is also absent from m2, which is the only family member that does not interact with Neur and fails to inhibit Notch when overexpressed. Deletion and point mutation analysis of Tom further demonstrates that motif 2 is important for Neur binding: (1) truncated versions of Tom lacking motif 2 did not interact with Neur in the two-hybrid assay; (2) internal deletion of this motif strongly impaired interaction in the two-hybrid assay; (3) alanine substitution of either 11 or 4 residues of motif 2 also impair interaction. It is concluded that motif 2 is important for Neur binding (Bardin, 2006).

Activation of DSL signaling by Neur is regulated by multiple mechanisms. A first level of regulation operates at the transcriptional level, both along the DV axis in the early embryo and within proneural clusters during imaginal development. A second level of regulation is seen during asymmetric division of the SOP with the unequal partitioning of Neur at mitosis. This study identifies a third level of regulation based on the inhibition of Neur by Brd family members. All Brd family members (with the exception of m2) interact in the yeast two-hybrid assay with the E3 ubiquitin ligase Neur. The overexpression of Brd genes specifically inhibits Neur-dependent Notch signaling events and leads to a defect in Dl endocytosis. Conversely, loss of the Brd-C that contains six out of the ten Brd genes results in ectopic Dl endocytosis and ectopic expression of the Notch target gene sim in the early embryo. Finally, physical interaction of Tom with Neur appears to inhibit the interaction of Neur with its substrate Dl. A model is proposed whereby proteins of the Brd family antagonize Neur-mediated Dl signaling by inhibiting the interaction of Dl with Neur (Bardin, 2006).

Precise positioning of the mesectoderm results from the integration of different activities that are more broadly distributed along the DV axis. The DV gradient of nuclear Dorsal is interpreted to establish large domains of gene expression. The twist gene is expressed in a large ventral territory that encompasses the mesoderm, whereas the expression of the snail gene becomes restricted to the mesoderm. Twist and Dorsal activate the expression of the sim gene whereas Snail represses it. Neur-dependent Dl signaling in the mesoderm is thought to further restrict sim expression to cells in direct contact with the mesoderm. The signaling activity of Dl is thought to be restricted to the mesoderm because its endocytosis is tightly restricted to the mesoderm in stage 5 embryos. While transcriptional regulation of neur in ventral cells likely contributes to this spatial regulation, it cannot on its own account for the mesoderm-specific regulation of Dl endocytosis. Indeed, high levels of transcripts are detected in ventral cells outside the mesoderm and low levels of transcripts are detected all around the embryo. This suggests that a posttranscriptional inhibitory mechanism exists to ensure that Neur is not active outside the mesoderm. This study shows that Brd proteins inhibit Neur-mediated Dl endocytosis and Notch signaling in nonmesodermal cells. It is also shown that ectopic expression of Tom inhibits the endocytosis of Dl in the mesoderm. This suggests that the repression of the expression of Brd-C genes in the mesoderm is important for Neur to be active in this tissue. Inhibition of Tom expression (and possibly of the other Brd-C genes) in the mesoderm depends on the mesoderm-specific repressor Snail. Accordingly, the ectopic expression of Brd genes in ventral cells of snail mutant embryos may explain the loss of Dl endocytosis and Notch activation that was previously observed in these embryos. It is therefore suggested that the Brd-C genes represent the hypothesized Snail target gene X proposed to act as a negative regulator of Notch signaling and Dl endocytosis (Morel, 2003). Thus, the sharp boundary of Snail expression appears to define the ventral limit of Brd family gene expression, hence the dorsal limit of Neur activity and Dl signaling. In summary, these data support a model whereby the Brd genes prevent ectopic Notch activation in the early embryo and contribute to DV patterning by restricting the mesectoderm territory to a single row of cells (Bardin, 2006).

The function of the Brd genes is probably not restricted to the early embryo. Indeed, several Brd genes are also strongly expressed during early neurogenesis in the embryo as well as in the proneural clusters of the eye, leg, and wing imaginal discs. Proneural cluster expression of the Brd genes may be important to restrict, in space and/or time, the activity of Neur during the process of SOP determination. While Neur appears to be primarily expressed in the presumptive SOP, there is also evidence that Neur may also be expressed at low levels in non-SOP cells. In particular, low-level expression of Neur in non-SOP cells is occasionally seen using neurP72Gal4. It is hypothesized that Brd may act to antagonize this low level of Neur activity in proneural cluster cells. Interestingly, the expression of the neur gene in SOPs is accompanied by the transcriptional repression of the gene by Su(H) in SOPs. The expression of other Brd family genes is excluded from SOPs, suggesting that they may also be repressed by Su(H). Conversely, the positive regulation of Brd gene expression by Notch in non-SOP cells correlates with a loss in Dl signaling activity in these cells. It is therefore speculated that the Brd genes contribute to amplify an initially weak difference in Dl signaling activity between presumptive SOP and non-SOP cells (Bardin, 2006).

The role proposed for the Brd genes in lateral inhibition remains to be investigated. It was found that the deletion of the Brd-C is largely embryonic lethal. However, a few homozygous Brd-C1 escaper flies are observed. These Brd-C1 flies show no detectable defects in bristle density. While this observation indicates that the Brd-C does not play an essential role in the process of SOP selection, the possibility remains that the and m4 genes act redundantly with genes of the Brd-C in this process (Bardin, 2006).

This study shows that all Brd family members (with the exception of m2) interact with Neur in the yeast two-hybrid assay. Interaction of Tom with Neur was further confirmed by coimmunoprecipitation experiments. Importantly, interaction of Tom with Neur correlates with a decrease in the amount of Dl immunoprecipitated by Neur, without affecting the levels of Neur and/or Dl. It is noted, however, that TomΔ2, which interacts weakly with Neur, can still inhibit interaction of Dl with Neur in this assay. The ability of Tom to decrease the Dl-Neur binding in this assay has led to a model whereby Brd family members antagonize Neur-mediated Dl signaling by inhibiting the Neur-Dl interaction. This model is consistent with the observations that overexpression of Tom has no effect on Neur protein levels. It is also consistent with the observation that Tom blocks the activity of NeurC701S. The latter may act in a dominant-negative manner by titrating DSL ligands. Accordingly, Tom could prevent NeurC701S from titrating DSL ligands. Similarly, the failure of Tom to suppress the wing phenotype induced by Mib1C1205S is consistent with the observation that Tom does not bind Mib1 and cannot, therefore, prevent Mib1C1205S from titrating DSL ligands. Whether Brd family members inhibit Neur by competing with Dl for overlapping binding sites remains to be investigated (Bardin, 2006).

These studies have focused on the interaction between Neur and a single Brd family member for the sake of consistency. Tom was chosen because (1) it includes all four conserved motifs present in the various Brd family members; (2) it is the Brd gene that aligns best with the single Anopheles Brd gene; (3) its overexpression gives a strong gain-of-function phenotype; and (4) it is expressed at high levels in stage 5 embryos. Whether all Brd family members similarly act by inhibiting the Neur-Dl interaction remains to be fully investigated. Because all Brd family members (with the exception of m2) have been shown to inhibit Neur-mediated Notch signaling, it is likely that all Brd family members similarly inhibit Neur. This in turn raises the question of the role of the two additional conserved motifs found at the C terminus of Ocho, Tom, mα, m4, and m6 that are also conserved in the single Bombyx and Anopheles Brd homologs (Bardin, 2006).

While Neur has homologs in vertebrates and Xenopus Neur has been suggested to regulate Dl signaling during early neurogenesis, no obvious homologs of the Brd genes are detectable in vertebrate sequenced genomes. This does not, however, exclude the possibility that vertebrate genes encoding Brd-like inhibitors exist. Indeed, motif 2 of Brd may be too short to reliably detect possible Brd homologs in vertebrate genomes by sequence alignments (Bardin, 2006).

This study has shown that Brd family members interact with Neur and block Neur-mediated Dl endocytosis. The activity of the Brd-C is required to spatially restrict Dl signaling along the DV axis in the early embryo (Bardin, 2006).

Dorsal-ventral pattern of Delta trafficking is established by a Snail-Tom-Neuralized pathway

The intracellular trafficking of the Notch ligand Delta plays an important role in the activation of the Notch pathway. This study addresses Snail-dependent regulation of Delta trafficking during the plasma membrane growth of the mesoderm in the Drosophila embryo. Delta is retained in endocytic vesicles in the mesoderm but expressed on the surface of the adjacent ectoderm. This trafficking pattern requires Neuralized. A protocol based on chromosomal deletion and microarray analysis has led to the identification of tom as the target of snail regulating Delta trafficking. Snail represses Tom expression in the mesoderm and thereby activates Delta trafficking. Overexpression of Tom abolishes Delta trafficking and signaling to the adjacent mesoectoderm. Loss of Tom produces mesoderm-type Delta trafficking in the entire blastoderm epithelium and an expansion of mesoectoderm gene expression. It is proposed that Tom antagonizes the activity of Neuralized and thus establishes a sharp mesoderm-mesoectoderm boundary of Notch signaling (De Renzis, 2006).

The Neuralized-dependent trafficking of Delta in the signal-sending cell is required for Notch activation in the receiving cell. Therefore, the activity of Neuralized must be tightly controlled between the signal-sending and signal-receiving cell in order to ensure the correct pattern of Delta trafficking and signal polarity. This study has followed the snail-mediated modulation of Delta trafficking during the cellularization of the Drosophila embryo. The concomitant formation of cell membranes and activation of zygotic transcription has offered some unique advantages for the experiments described in this work. Most importantly, the growth of the plasma membrane is timed with the zygotic expression of snail and neuralized and thus allows a precise staging protocol (De Renzis, 2006).

The experiments demonstrate that snail regulates the mesoderm-specific trafficking of Delta by repressing the expression of tom, and presumably of the other brd genes. In the ectoderm, where the brd class genes are expressed and snail is not, Delta and Notch Extra-Cellular Domain (NECD) have a predominantly cell surface localization. Obvious endocytic vesicles containing these proteins do not accumulate in ectodermal cells. Such vesicles do form in the mesoderm where Snail represses tom expression, and in tom−/− embryos, where the NECD-Delta vesicles extend into the ectoderm. The vesicular trafficking of Delta and NECD characteristic of mesodermal cells requires the ubiquitin ligase Neuralized. Overexpression of Tom recapitulates the loss of function phenotype of neuralized, consistent with the view that Tom may normally function by opposing the role of Neuralized in Delta trafficking. Neuralized expression is dynamic and extends to the lateral region of the embryo, beyond the mesoderm-ectoderm boundary. Thus, on its own it cannot explain the restriction of vesicles to the mesoderm. It is proposed instead that Tom creates a functional boundary for Neuralized by suppressing its activity in the ectoderm. In agreement with this model, in tom−/− embryos the expression of the mesoectoderm gene sim is extended dorsally (De Renzis, 2006).

The expression of sim is regulated by the maternal Dorsal nuclear gradient that directly or indirectly specifies all ventral cell fates. The mechanisms that precisely position sim expression to one row of cells, however, are not completely understood. sim can respond to the Dorsal gradient and can in principle be expressed in the entire ventral region of the embryo. Recent studies suggest that Notch signaling restricts the expression of sim to the mesoectoderm by relieving Suppressor of Hairless [Su(H)]-mediated repression of sim. Su(H) is uniformly distributed throughout the early embryo and represses sim expression in the ectoderm (De Renzis, 2006).

The precision of sim expression and its one cell diameter reflect the bias that zygotic expression of tom introduces on maternal Notch signaling in that region of the embryo. If the mesoderm cells that do not express Tom are the only cells that retain Neuralized activity and can internalize Delta ligand, they might be the only cells capable of sending signal. The snail repression will allow sim expression outside the mesoderm, and thus only those cells could be effective signal recipients. According to this model, the last snail-expressing cell of the mesoderm becomes the signal-sending cell and its immediate dorsal neighbor becomes the signal-receiving cell; i.e., the mesoectoderm. In the absence of Tom, the dynamic expression of Neuralized, which extends dorsally, would make more cells competent to send Notch signal. Indeed, the dorsal expression of sim is more pronounced at the end of mesoderm invagination at the time when the expression of Neuralized has extended to the neuroectoderm (De Renzis, 2006).

The molecular mechanisms by which Tom functions are most likely related to its interaction with Neuralized; this has been demonstrated in a yeast-two hybrid genomic screening (Giot, 2003). Tom may inhibit the ubiquitin ligase activity of Neuralized or it could compete for the interaction between Delta and Neuralized. Future work will be necessary to discriminate between these two possibilities. It will also be important to test the activity of the other Brd family members in the regulation of Neuralized activity. At least eight bearded-like genes (m2, m4, m6, , bob, brd, tom, and ocho) have been identified in the Drosophila genome. An interesting possibility is that different genes in this family may have different effects on Delta trafficking. Any difference may provide important clues into the regulation of the Notch pathway (De Renzis, 2006).

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

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