BIOLOGICAL OVERVIEW

Strawberry notch is a nuclear protein that functions downstream of Notch. The loss of sno expression at the margin of the developing wing results in the loss of expression of wingless, vestigial, cut and Enhancer of split-m8, the latter being a member of the Enhancer of split complex (Majumdar, 1997). In regulating these genes, Sno functions in close cooperation with Suppressor of Hairless, a gene directly involved in the transduction of the Wingless signal, and in cooperation with Hairless, which physically interacts with Su(H) preventing Su(H) from binding to DNA, and thus counteracting Notch signaling (Majumdar, 1997 and Brou, 1994).

Genetic studies of sno reveal an interesting dichotomy with regard to Notch function: Notch's lateral inhibition function has been well documented, but Notch signaling in gene induction, involving sno, appears to be regulated differently. Phenotypes of embryos with null mutations in sno show no evidence of neural hypertrophy, the phenotype that evidences Notch's lateral inhibition function. Because there is a considerable maternal contribution of Sno in the embryo, lack of a neural hypertrophy phenotype in sno mutants could be explained by continued presence of maternal Sno during embryogenesis. Therefore, in order to investigate the possible role of Sno in lateral inhibition, the pupal stage, taking place long after disappearance of maternally contributed mRNAs became the focus for investigation (Majumdar, 1997).

Considerable effort has gone into examining whether Sno is involved in lateral inhibition. Each microchaete sensillum on the adult notum, derived from the wing imaginal disc, arises during pupal development from a sensory organ precursor (SOP) cell. A single SOP is specified from within a group of equipotential cells called the proneural equivalence group by means of Notch mediated lateral signaling. The absence of Notch signaling (maintaining flies with temperature sensitive Notch at non-permissive temperatures) results in multiple SOPs developing from each field of competent cells. Normally only one SOP is present. Under identical conditions, flies mutant in sno do not show any overproduction of microchaetes, suggesting that lateral signaling during SOP specification is not affected in sno mutants. The temperature used to inactivate sno is clearly nonpermissive for all other sno phenotypes, and in fact, causes lethality at later pupal stages. In particular, such flies loose expression of wingless at the wing margin under the same conditions that fail to produce a neurogenic (lateral inhibition) phenotype. It thus appears that Sno is not involved in the lateral signaling during SOP specification under the same conditions in which sno mutants loose expression of wingless in the wing margin. Thus Sno is involved in induction of wingless but not in lateral inhibition (Majumdar, 1997).

Although the mutant conditions used were sufficient to inactivate sno function in many tissues, one could argue that the threshold levels for lateral signals are lower than for induction signals, thus explaining the lack of a lateral inhibition phenotype. To rule out this possibility, somatic clones of null alleles of sno were generated in the eye. These clones produced one of the phenotypes ascribed to sno. Mystery cells (cells that are left out of the differentiation process and that ultimately are normally destroyed by programmed cell death) are recruited to ommatidial clusters as outer neurons. sno mutants show no such recruitment. In contrast, the sno clone does not show hypertropy of neural development behind the morphogenic furrow as seen with loss of Notch function. It has thus been concluded that sno does not function in lateral signaling within pro-neural equivalence groups, but rather Sno participates only in inductive Notch signaling events (Majumdar, 1997).

It has been suggested that the selective activation of Sno in signal-receiving cells may confer the specificity for inductive versus lateral signaling. A counterexample related to Sno function in this regard is Neuralized, which may function specifically in lateral signaling much as Sno does in inductive pathways, since neuralized is not expressed along the D/V boundary of the wing and does not interact genetically with sno at the wing margin (Coyle-Thompson, 1993 and Majumdar, 1997).

In C. elegans, two separate Notch receptor homologs, Lin-12 and Glp-1, function in separate developmental pathways. Glp-1 is involved in inductive signaling both in the gonad and in the early embryo, whereas Lin-12 function is largely restricted to lateral signaling during different stages of development. However, even in the worm, the specificity of the response of the two sets of receptors does not seem to depend on the receptors themselves, because they can be made to function interchangeably in an inductive (versus a lateral) signaling pathway (Fitzgerald, 1993). Therefore, in C. elegans, as in Drosophila, it is possible that a differential response to common signal transduction pathways might be brought about by downstream molecules similar to Sno (Majumdar, 1997).

The Notch and Epidermal growth factor receptor (Egfr) pathways both regulate proliferation and differentiation, and the cellular response to each is often influenced by the other. A mechanism is described that links them in a sequential fashion, in the developing compound eye of Drosophila. Egfr activation induces photoreceptor (R cell) differentiation and promotes R cell expression of Delta. This Notch ligand then induces neighboring cells to become nonneuronal cone cells. ebi and strawberry notch (sno) regulate Egfr-dependent Delta transcription by antagonizing a repressor function of Suppressor of Hairless [Su(H)]. Sno binds to Su(H), and Ebi, an F-box/WD40 protein, forms a complex with Su(H) and the corepressor Smrter. Egfr-activated transcriptional derepression requires ebi and sno, is proteasome-dependent, and correlates with the translocation of Smrter to the cytoplasm (Tsuda, 2002).

The Notch signaling pathway plays multiple roles in eye development. At the morphogenetic furrow, the proneural protein Atonal facilitates the expression of Dl in the R8 cell. The first step of ommatidial assembly involves lateral inhibition between equivalent cells, but successive steps are inductive, arising from an already differentiated cell to its uncommitted neighbors. The Notch pathway is involved in the regulation of both of these processes. Similarly, the Egfr ligand, Spi, expressed in R8, activates the receptor in neighbors allowing them to assume their respective R1–R7 cell fates. Subsequently, these R cells express Spi, and as described in this study, they also express Dl in response to Egfr activation. The cone cells receive an Egfr signal and a Notch signal from the R cells and this combination is critical for the assumption of their fate. Later, after their fate is determined, these cone cells, too, will express Delta, which is important for pigment cell induction. Presumably, the level of the Egfr signal rises in the cone cells with time, and as a threshold of Egfr activation is surpassed, the proteasome mediated arm of the pathway becomes effective causing derepression of Su(H) and expression of functional levels of Dl sufficient for pigment cell development. Thus, a temporally and spatially positioned combination of parallel and sequential Egfr/Notch signals is important for the successive induction of cell types in the eye (Tsuda, 2002).

An interesting interplay between Egfr and Notch pathways is also seen during vulval induction in C. elegans. Cells that are close to the anchor cell assume the primary developmental fate, while those farther away become secondary cells. The development of the secondary cell fate shows many similarities with cone cell development. Both secondary and cone cells primarily require high levels of Notch signal and a low-level activation of the Egfr signaling pathway. Genetic studies support one of two alternative models for the development of the secondary cell fate. In the first model, the graded activation of Egfr (Let23) mediated by the expression of its ligand Lin3 in the anchor cell and lateral Notch (Lin12) signaling imparts a secondary cell fate. Alternatively, the signal mediated by Lin3 is required for the specification of the primary cell, which in turn induces secondary cells through the Notch pathway. The latter model is similar to the sequential activation mechanism describe in this study for cone cell development. It will be interesting to determine if in C. elegans, the Egfr (Let23) pathway activates an as yet unidentified Notch (Lin12) ligand in primary cells that is then used to induce secondary cell fate (Tsuda, 2002 and references therein).

Evidence from mammalian systems has suggested that CBF1, the mammalian homolog of Su(H), is a component of a large repressor complex. The activation function of CBF1 results from a displacement of repressive components (such as HDAC) by the intracellular domain of Notch which converts Su(H) into a transcriptional activator. Genetic analysis of the embryonic midline and the pupal bristle complexes in Drosophila have also supported a switch from Su(H)-mediated repression to activation. A second mechanism for relieving Su(H) mediated repression is through Sno, Ebi, and the Egfr pathway. In response to the Egfr signal, Ebi, an F-box protein, presumably causes a proteasome-mediated degradation of an unknown component of the Su(H) inhibitory complex. Mammalian TBL1 (Ebi) can function downstream of the tumor suppressor gene, p53, in the degradation of the ß-catenin protein in a novel ubiquitin-dependent degradation pathway involving Siah, the mammalian homolog of the Drosophila Sina protein. Similarly, Drosophila Ebi can also act in combination with Sina to degrade protein targets. More generally, phosphorylation by MAPK downstream of RTK pathways is known to trigger proteasome-mediated degradation of target proteins. In addition to Ebi, a core component of the proteasome, encoded by l(3)73Ai gene, is also important for expression of Dl. The simplest model is that in response to Egfr signaling, one or more of the many components in the large Su(H)/SMRTER repression complex becomes a target of a proteasome-mediated degradation process (Tsuda, 2002).

The studies presented here also show that the corepressor SMRTER is redistributed from the nucleus to the cytoplasm in an Egfr/Sno/Ebi dependent manner. These results are in complete agreement with the role of the corresponding mammalian protein SMRT in its function as a repressor. Like Su(H), nuclear hormone receptors such as retinoic acid receptor and thyroid hormone receptor can function as both repressors and activators. SMRT has been shown to be phosphorylated in response to an RTK signal. This leads to translocation of SMRT out of the nucleus. Thus, steroid hormone receptors lose their ability to repress but not activate transcription. In an in vivo example, the Egfr/Sno/Ebi pathway promotes the dissociation of the Su(H)/SMRTER repressor complex and causes the nuclear export of SMRTER. As a result, target genes such as Dl are derepressed (Tsuda, 2002).

Notch signaling can take place between cells that are equivalent at the time the signal initiates, or it can occur between a signaling cell that is different from the cell receiving the signal. Traditionally, the first kind of process has been referred to as lateral inhibitory Notch signaling and the second as an inductive Notch pathway. These studies suggest that the fundamental difference between these two processes is not due to differences in molecular components of the pathway downstream from activated Notch, but rather due to the mechanism that controls the expression of the ligand, Dl. For lateral inhibitory Notch pathways, a mechanism involving a feedback loop and proneural genes is at the core of Dl/Notch regulation. Starting with an equipotent group, an asymmetric signaling system is created, in which the signaling cell expressing high levels of Dl, assumes a differentiated fate and prevents its neighbors from adopting an identical fate. All available evidence suggests that the Egfr pathway, Sno, and Ebi do not control Dl expression in such lateral inhibitory processes mediated by Notch. In contrast, this study shows that in inductive processes controlled by Notch signaling, Dl expression is controlled by Egfr, Ebi, and Sno and apparently not by proneural genes. For example, no known proneural gene (Ac/sc, amos, or atonal) is expressed in R cells that contact the cone cells (i.e., R1-R7) and express Dl. This is also true for cells at the dorsoventral boundary of the wing disc where Notch signaling directly activates vestigial expression through Su(H) binding to the enhancer and in the mesectodermal cells of stage 6 embryos where the Notch pathway has been implicated in controlling the expression of single minded at the midline. Instead, all of these cells in the eye, wing, and embryo receive an Egfr signal that likely controls Dl expression. Indeed, the late expression of Dl in R cells does not involve feedback from the cone cells but instead involves the derepression of Dl expression in a Notch-independent manner. This is different from the early expression of Dl that is required for the selection of R8 cells at the furrow through a lateral inhibitory signal (Tsuda, 2002).

This study highlights the function of two unusual proteins, Sno and Ebi, in controlling the expression of Dl. Mammalian Ebi (TBL1) interacts with a SMRT/HDAC complex as also supported by this study in Drosophila. There are two human and three mouse genes similar to Sno identified by genome projects. The function of the mammalian Sno proteins is unknown. Whether the mammalian proteins also function upstream of the Notch pathway, as they do in Drosophila, remains to be established. Given the conservation of developmental pathways between Drosophila and mammals, this may not be an unreasonable expectation (Tsuda, 2002).

GENE STRUCTURE

Bases in 5' UTR - 319

Exons - 5

Bases in 3' UTR - 780

PROTEIN STRUCTURE

Amino Acids - 1547

Structural Domains

The Sno protein contains no similarities to previously described proteins. However two nuclear localization signals and two acidic regions are found, suggesting that Sno plays a role in transcriptional activation. The Sno sequence shows a high degree of similarity to a C. elegans sequence. The best overlap is in two blocks of residues, 318-721 and 997-1205 from the Drosophila sequence. These two regions show 66% and 71% identity, respectively, to the C. elegans sequence. The second acidic region of the fly protein is conserved. A portion of the fly Sno protein is also conserved in two human cDNAs and a mouse sequence (Majumdar, 1997).

Notch and Hippo signaling converge on Strawberry Notch 1 (Sbno1) to synergistically activate Cdx2 during specification of the trophectoderm

The first binary cell fate decision occurs at the morula stage and gives rise to two distinct types of cells that constitute the trophectoderm (TE) and inner cell mass (ICM). The cell fate determinant, Cdx2 (Drosophila homolog: Caudal), is induced in TE cells and plays an essential role in their differentiation and maintenance. Notch and Hippo signaling cascades are assumed to converge onto regulatory elements of Cdx2, however, the underlying molecular mechanisms are largely unknown. This study show involvement of Strawberry Notch1 (Sbno1; Drosophila homolog Sno), a novel chromatin factor of the helicase superfamily 2, during preimplantation development. Sbno1 knockout embryos die at the preimplantation stage without forming a blastocoel, and Cdx2 is not turned on even though both Yap (see Drosophila Yorkie) and Tead4 (see Drosophila Scalloped) reside normally in nuclei. Accordingly, Sbno1 acts on the trophectoderm-enhancer (TEE) of Cdx2, ensuring its robust and synergistic activation by the Yap/Tead4 and NICD/Rbpj complexes. Interestingly, this synergism is enhanced when cells are mechanically stretched, which might reflect that TE cells are continuously stretched by the expanding ICM and blastocoel cavity. In addition, the histone chaperone, FACT (FAcilitates Chromatin Transcription) physically interacts with Sbno1. These data provide new evidence on TE specification, highlighting unexpected but essential functions of the highly conserved chromatin factor, Sbno1 (Watanabe, 2017).

date revised: 15 January 2003  

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