BIOLOGICAL OVERVIEW

Enhancer of split complex [E(spl)-C] genes are the target of the Notch pathway. Enhancer of split is one of seven genes of the E(spl)-C. Signals from the Notch receptor are carried from the surface of the cell to the nucleus, by the dual function protein Suppressor of Hairless. This protein interacts with Notch at the cell surface as well as acting as a transcription factor involved in the nuclear regulation of Enhancer of split. This cascade of events, beginning with Notch signaling and leading to the eventual involvement of E(spl)-C genes, constitutes the neurogenic pathway in the fly (Lecourtois, 1995 and Bailey, 1995).

Neurogenesis takes place in the ventral neuroectoderm, which gives rise to the ventral nerve cord, commonly called the central nervous system, and in the procephalic neurogenic region, the part of the head that gives rise to the brain. The process starts in the ventral neuroectoderm with the proneural cluster, a group of cells consisting of up to 16 cells per cluster. Usually, only one cell from each proneural cluster becomes committed to the neural fate and delaminates, while the others revert to an ectodermal status. E(spl) and the other six genes of the E(spl)-C act to shut down neurogenesis, particularly in those cells that will remain part of the ectoderm. Thus E(SPL) protein acts as a repressor.

Loss of E(spl)-C function results in a neural hyperplasia (too many cells), and ultimately, in cell death. E(spl) and other genes of the E(spl)-C counteract the tendency of neural precursors to divide, and thereby ensure the integrity of the neural ectoderm that gives rise to neural progenitors.

In addition to Suppressor of Hairless, several other genes regulate E(spl) and other E(spl)-C genes. achaete, scute, and ventral nervous system defective/NK2 are postive regulators, and E(spl) itself is involved in an autoregulatory loop.

Only groucho and E(spl) among the genes of E(spl)-C show visible phenotypes when mutant. E-splwas named for the characteristic that first identified it: the ability to enhance split mutations. split however, is just another name for Notch (Welshons, 1956).

Enhancer of split complex genes regulate Delta by acting through achaete-scute complex genes. Mutation of E(spl)-C genes or groucho, like Notch or Delta mutants cause an overproduction of sensory organs precursors at the expense of epidermis. Like mutant Notch cells. Cells mutant for E(spl)-C genes or groucho inhibit neighouring wild-type cells destined for neural fate, thus causing them to adopt the epidermal fate. This inhibition requires the genes of the achaete-scute complex, which would be in a more active state in cells mutant for E(spl)-C genes or groucho. This active state would elevate Delta transcription, repressing the neural fate of neighboring cells. Thus there is a regulatory loop between surface Notch via the E(SPL)-C and the AS-C that normally represses Delta, allowing for the adoption of the neural fate in adjacent cells (Heitzler, 1996).

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 46^th (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).

Regulation of Notch output dynamics via specific E(spl)-HLH factors during bristle patterning in Drosophila

The stereotyped arrangement of sensory bristles on the adult fly thorax arises from a self-organized process, in which inhibitory Notch signaling both delimits proneural stripes and singles out sensory organ precursor cells (SOPs). A dynamic balance between proneural factors and Enhancer of split-HLH (E(spl)-HLH) Notch targets underlies patterning, but how this is regulated is unclear. This study identified two classes of E(spl)-HLH factors, whose expression both precedes and delimits proneural activity, and is dependent on proneural activity and required for proper SOP spacing within the stripes, respectively. These two classes are partially redundant, since a member of the second class, that is normally cross-repressed by members of the first class, can functionally compensate for their absence. The regulation of specific E(spl)-HLH genes by proneural factors amplifies the response to Notch as SOPs are being selected, contributing to patterning dynamics in the notum, and likely operates in other developmental contexts (Couturier, 2019).

Pattern formation is a central question in developmental biology. Patterns of differentiated cells that are invariant across individuals can be observed in many species. These can be generated by interpreting a fixed pre-pattern or via self-organization, possibly guided by fixed initial conditions. The patterns of sensory organs in Drosophila are model systems to study how simple patterns dynamically emerge during development. On the dorsal thorax, or notum, macrochaetae are found at fixed positions and microchaetae are regularly distributed in five dorso-central rows in each hemi-notum. As each of these sensory bristles develops from a single sensory organ precursor cell (SOP), their pattern in the adult results from the pattern of SOPs in imaginal tissues in late third instar larvae and early pupae. SOPs emerge from groups of cells that express one or more transcriptional activators of the bHLH proneural family, e.g., Achaete (Ac) and Scute (Sc). These factors confer these cells with the ability to become SOPs and these groups of cells are known as proneural clusters. Thus, the position-specific expression of Ac and Sc determine where sensory bristles can develop. Two models have been proposed to explain how proneural clusters develop. In a first model, positional cues govern early proneural activity, and thereby the stereotyped layout of sensory organs whereas inhibitory Notch signaling acts downstream of proneural activity to select SOPs within each proneural cluster. This model applies well for the macrochateae. In a second model, it is instead Notch activity that negatively defines where proneural activity can emerge, and self-organization involving cell-cell interactions mediated by Notch directs patterning dynamics at the tissue scale. In this model, the role of positional cues is limited to defining the initial and/or boundary conditions that guide self-organized Notch dynamics. This model is proposed to apply for the five rows of microchaetae in the dorsal-central notum, which arise from a series of proneural stripes that emerge in a defined sequence (Couturier, 2019).

Once proneural clusters or stripes have formed, inhibitory cell-cell interactions mediated by Notch restrict the potential to become an SOP to one or a few cells per cluster (or stripe). Notch inhibits the competence to become neural via the E(spl)-HLH family of transcriptional repressors which act redundantly to antagonize the activity and expression of Ac and Sc. Therefore, adoption of the SOP fate depends on a balance between the activity of Ac and Sc, acting synergistically with Senseless (Sens), and the anti-proneural activity of the E(spl)-HLH proteins. How this balance is dynamically regulated during patterning in the notum is not clear, in part because the dynamic expression of individual E(spl)-HLH factors is not known. While all seven E(spl)-HLH genes are directly regulated by Notch, each of these genes has, however, a unique expression pattern. The transcriptional response of the E(spl)-HLH genes to Notch is therefore context-specific, and spatially restricted factors, including Ac and Sc, appear to cooperate with Notch for their regulation in embryos and imaginal tissues. Thus, whether and how different E(spl)-HLH factors contribute to the evolution of the proneural pattern and the emergence of isolated SOPs remains to be studied (Couturier, 2019).

This study examined the role of Notch signaling in early stripe patterning and characterized the expression and function of the different E(spl)-HLH factors in the developing notum. Using reporters for each of the seven E(spl)-HLH proteins, it was found that a subset of E(spl)-HLH factors are expressed early, prior to the onset of proneural activity, and that additional E(spl)-HLH factors become expressed late, in a proneural-dependent manner. Early-onset factors define where the first stripes emerge while late-onset factors contribute to SOP selection. It is proposed that the regulation of specific E(spl)-HLH factors by Ac and Sc promotes mutual inhibition through a proneural-dependent increase in the number of E(spl)-HLH genes responding to Notch in cells with intermediate levels of Ac and Sc (Couturier, 2019).

An early, widespread and evolutionarily conserved response of the genome to Notch activation is the CSL-dependent transcription of the HES family genes. In Drosophila, the seven HES family genes encoded by the E(spl)-C act redundantly and the relative contribution of individual E(spl)-HLH factors to the overall Notch output has remained unclear. For this study a complete set of GFP-tagged reporters were generated, and the expression dynamics of all E(spl)-HLH factors were examined in the developing notum. m3 and mβ are expressed early in response to Mib1-dependent Notch signaling, and additional E(spl)-HLH factors, notably mδ, m7, and m8, become expressed later, once proneural stripes are established and in a proneural-dependent manner. Early-onset factors appeared to mediate the negative template activity of Notch for early stripe patterning, while late-onset factors, notably m7 and m8, are essential to reach sufficient Notch signaling output for the proper spacing of sensory bristles. Thus, different E(spl)-HLH factors contribute at distinct steps of this patterning process and the regulation of specific E(spl)-HLH genes by Ac and Sc plays an important role by raising the level of E(spl)-HLH activity, produced in response to Notch, in groups of cells progressing towards the SOP fate. This upregulation is transient, and once proneural stripes have resolved, the same early-onset factors that mediate stripe patterning are expressed in non-SOP cells to lock down their fate (Couturier, 2019).

The regulation of the E(spl)-C genes by a dual Notch/proneural input has been well studied before. This regulation was previously interpreted to suggest that Ac and Sc set up the initial conditions for a regulatory feed-back loop operating between proneural cluster cells. In this model, Ac and Sc initiate the conditions for both signaling, via the regulation of the Dl and neur genes, and responding to Notch, via the regulation of the E(spl)-C genes. Detailed analysis of the expression, regulation and function of the E(spl)-HLH factors did not support this model. Indeed, the proneural-independent expression of m3 and mβ, downstream of Mib1-dependent Notch signaling, appeared to provide the initial conditions for patterning. A different model is proposed different in this paper, whereby the proneural-dependent regulation of specific E(spl)-HLH factors serves to modulate mutual inhibition within the proneural stripes during SOP selection, and thereby to shape the dynamics of patterning. Cells with intermediate levels of Ac and Sc, because they activate an increasing number of Notch-responsive E(spl)-HLH genes, cannot evade mutual inhibition as readily as they would otherwise. Such a regulatory logic may favor the robust emergence of regularly spaced sensory organs (Couturier, 2019).

A role for the modulation of mutual inhibition was anticipated by an abstract mathematical model that recapitulates the temporal and spatial dynamics of fate patterning in the notum, and the current findings suggest a molecular basis for several features of the model's dynamics. In brief, this simple model represents the state of each cell by a single variable, u. This variable varies in time as a function of an inhibitory signal, s, representing the level of Notch ligands to which the cell is exposed, produced by other cells according to their own state u. Cells in the model have bistable dynamics, tending to one of two stable cell states, a high u/low s state (SOP fate; high proneural activity and low inhibitory signal) and a low u/high s state (non-SOP fate; low proneural activity and high inhibitory signal). Given appropriate initial conditions, the model recapitulates the sequential emergence of proneural stripes and their resolution into SOP rows. In simulations of this mathematical model, the balance between activation and inhibition is such that cells located at the center of the proneural stripes and progressing towards the SOP fate show increasing levels of both proneural activity and inhibitory signal (that is, of u and s). Interestingly, these simulations predicted that, once a proneural stripe emerges, the inhibitory signal is strongest in cells at the center of the stripe. This prediction was, however, not verified using GFPm3 as a Notch activity reporter: high levels of GFPm3 were observed in cells flanking the proneural stripes, not at the center of the stripes where SOPs develop. While cis-inhibition of Notch by Dl could account for a discrepancy between the inhibitory signal in the model (representing ligand levels) and Notch activity itself, detailed analysis of E(spl)-HLH expression suggests a different explanation: this particular Notch target, m3, does not fully reflect the in vivo activity of Notch, and other E(spl)-HLH factors, notably mδ, m7, and m8, contribute to the Notch output, particularly at the center of the proneural stripes. The contribution of cis-inhibition, if any, may thus be limited to emergent SOPs, which exhibit the highest levels of proneural factors and low levels of all E(spl)-HLH factors. The model further tied a gradual narrowing of neural competence, as observed experimentally, to a progressive increase in the strength of mutual inhibition. While this may result from a modulation of ligand activity, e.g., by Neur, in signal-sending cells, the findings identify another contribution, from the regulation of Notch target expression in receiving cells (Couturier, 2019).

This analysis also raises additional questions about how Notch and Ac/Sc regulate gene expression dynamics in the notum. In particular, two observations deserve consideration. First, how is it that the late-onset genes (mδ, m7, m8 etc.) do not respond to Notch signaling prior to the proneural onset? Second, why are m3 and mβ are not maximally expressed at the center of the stripes, where inhibitory signaling is predicted to be maximal? Several models can be proposed. In a first accessibility model (see Simulation of a mathematical model for patterning in the notum), only a subset of the CSL-binding sites is accessible for binding by CSL/NICD complexes in the absence of Ac and Sc, so that only a fraction of the E(spl)-HLH genes, i.e., mβ and m3, is activated by Notch; upon expression of Ac and Sc, additional binding sites become accessible, possibly through a change in chromatin structure induced by Ac and Sc, resulting in the expression of additional E(spl)-HLH genes. In support of this model, the proneural factor Ascl1 was shown to bind both closed and open chromatin in mouse neural progenitors, and binding to closed chromatin appeared to promote accessibility. In a second cooperativity model, early-onset genes contain high-affinity CSL binding sites, whereas late-onset genes have low-affinity CSL binding sites, so that only early-onset genes, i.e., m3 and mβ, respond to a Notch-only input; but the low-affinity CSL binding sites of the late-onset genes would be located close to E-boxes, such that Ac and Sc promote cooperative binding, hence gene expression. While these two models could explain the temporal sequence of gene activation, they do not, however, explain why m3 and mβ are not maximally expressed at the center of the stripes. Two possible mechanisms might account for this observation. One possibility is that the nuclear concentration of NICD is limiting such that not all CSL binding sites can be occupied, even when Notch signaling is maximal. If so, binding sites would effectively compete for the binding of CSL/NICD. If CSL/NICD complexes preferentially bind the regulatory sites of late-onset genes, this should then result in lower levels of m3 and mβ expression when and where these sites become accessible (accessibility model) or bound by Ac and Sc (cooperativity model). A second possibility is that the expression of the m3 and mβ genes is inhibited by late-onset E(spl)-HLH factors. Further studies will address these different models (Couturier, 2019).

Auto-repression and cross-repression is seen within the HES gene family in vertebrates. By contrast, self/cross-inhibition by E(spl)-HLH factors had not been observed in Drosophila, prior to this study. This study showed that the mδ gene is de-repressed in mβ m3 double mutant pupae, and ectopic expression in mutant nota appeared strikingly similar to those of the mβ and m3 genes in wild-type pupae. This finding may help resolve a long-held paradox which is that E(spl)-HLH genes have very distinct expression yet are functionally redundant. Obviously, ectopic expression of one (or several) factor upon loss of one (or several) others would account for redundancy despite specificity in expression (Couturier, 2019).

This analysis provides no evidence for functional specificity at the molecular level amongst the E(spl)-HLH factors. For instance, m3 and mβ appeared to be functionally replaced by mδ. Similarly, SOP selection could be achieved, at least to some extent, by m7 and m8 alone, as well as by the other five factors, at the exclusion of m7 and m8. Thus, non-overlapping sets of E(spl)-HLH factors can provide a proper Notch output for stripe patterning and SOP selection. These observations, together with earlier findings, indicate that the E(spl)-HLH factors have very similar molecular activities. Thus, the proneural-dependent expression of additional E(spl)-HLH factors may simply result in a global increase of an anti-proneural activity that would be provided by any of these factors. Therefore, describing Notch output dynamics and understanding its regulatory logic in a given context may require the analysis of all E(spl)-HLH factors that collectively contribute to this output. The tools generated in this study will help to achieve this (Couturier, 2019).

The patterning logic uncovered in this study may be of general relevance. While earlier studies viewed SOP selection in larvae as a multi-step process of proneural cluster resolution, the current data suggest that the progressive transition from mutual inhibition among proneural cells to lateral inhibition from SOPs is dynamically shaped by the regulation of a specific subset of E(spl)-HLH factors by Ac and Sc, with no need for specific mechanisms to restrict competence to a subgroup of proneural cells. In the adult fly gut, a subset of E(spl)-HLH genes, including mδ, m7, and m8, are also regulated by Sc and this regulation may be functionally relevant as Sc is a key cell fate regulator within the Intestinal Stem Cell (ISC) lineage. Interestingly, ISCs that are in a low Sc state may respond to Notch via mβ and m3 factors, which may suffice to remain in a low Sc state, whereas ISCs that are in a high Sc state may revert to a low Sc state in response to a stronger Notch output that would be produced in part through the mδ, m7, and m8 factors. Thus, the regulatory logic unraveled by this study may regulate cell fate within the ISC lineage (Couturier, 2019).

The regulation of the HES family genes by a dual Notch/proneural input is evolutionarily conserved. In Xenopus, an enhancer integrates the Notch and proneural inputs to regulate the expression of two Hes5-like genes. In the vertebrate brain, Ascl1 regulates the expression of Hey1, a direct Notch3 target. In this context, it is interesting to note that neural stem cells cycle between a quiescence state, regulated by high Notch3 activity and an activated state, also involving Notch3 but in the context of Ascl1 expression, a combination that might lead to expression of different HES family genes. Actually, in numerous contexts in development, Notch receptor activation intersects with the expression of proneural transcription factors. Thus, the regulatory logic uncovered here for the patterning of sensory organs, involving a proneural-dependent increase in E(spl)-HLH gene expression as a mean to upregulate the Notch output, may similarly operate in mammals (Couturier, 2019).

PROTEIN STRUCTURE

Amino Acids - 179

Structural Domains

The E(SPL) protein and each of the other bHLH genes are highly homologous to one another, especially the bHLH domain confined to the N-terminal half of the protein (Deladakis, 1992 and Knust, 1987).

To gain insight into the structural requirements for E(spl) function, a large number of deletion variants were expressed in transgenic flies. Three protein domains were identified as essential for suppression of bristle development: the carboxyl-terminal tetrapeptide WRPW, the region comprising the putative helix III and helix IV, and the region between helix IV and the WRPW motif. The bHLH domain is located in the N-terminal third of the protein; helix III and helix IV motifs are located C-terminal to the bHLH domain near the center of the protein and the WRPW motif is located in the C-terminus. Lack of either the basic helix-loop-helix domain, or helix III or IV only partially inhibits the suppressor activity of the protein. Truncated variants that lack all the regions carboxyl-terminal to helix IV elicit the development of additional neural progenitors, and thus act as dominant-negative variants. Moreover, the proline residue in WRPW is essential because its alteration to leucine results in a nonfunctional protein. A similar null hairy allele is associated with a proline to leucine exchange in its WRPW domain. All these results suggest that E(spl) suppresses neural development by direct interaction with other proteins, such as Groucho and the proneural proteins via the WRPW motif (Giebel, 1997).

date revised: 22 November 2022 

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