Notch signaling and Notch signaling modifiers

Originally discovered nearly a century ago, the Notch signaling pathway is critical for virtually all developmental programs and modulates an astounding variety of pathogenic processes. The DSL (Delta, Serrate, LAG-2 family) proteins have long been considered canonical activators of the core Notch pathway. More recently, a wide and expanding network of non-canonical extracellular factors has also been shown to modulate Notch signaling, conferring newly appreciated complexity to this evolutionarily conserved signal transduction system. Here, I review current concepts in Notch signaling, with a focus on work from the last decade elucidating novel extracellular proteins that up- or down-regulate signal potency.     

Stomaching Notch

The self‐renewal and differentiation of tissue stem cells must be tightly controlled. Unrestrained self‐renewal leads to over‐proliferation of stem cells, which may cause tumor formation, while uncontrolled differentiation leads to depletion of the stem cell pool. In this issue of The EMBO Journal, Demitrack et al (2015) show that the Notch pathway is a key regulator of Lgr5 antral stem cell self‐renewal and differentiation. Notch signaling controls the proliferation and differentiation of stem cells as well as gastric tissue growth, while uncontrolled Notch activity in stem cells leads to polyp formation.     

Notch2: a second mammalian Notch gene.

Notch is a cell surface receptor that mediates a wide variety of cellular interactions that specify cell fate during Drosophila development. Recently, homologs of Drosophila Notch have been isolated from Xenopus, human and rat, and the expression patterns of these vertebrate proteins suggest that they may be functionally analogous to their Drosophila counterpart. We have now identified a second rat gene that exhibits substantial nucleic and amino acid sequence identity to Drosophila Notch. This gene, designated Notch2, encodes a protein that contains all the structural motifs characteristic of a Notch protein. Thus, mammals differ from Drosophila in having more than one Notch gene. Northern and in situ hybridisation analyses in the developing and adult rat identify distinct spatial and temporal patterns of expression for Notch1 and Notch2, indicating that these genes are not redundant. These results suggest that the great diversity of cell-fate decisions regulated by Notch in Drosophila may be further expanded in vertebrates by the activation of distinct Notch proteins.     

Temporal Notch activation through Notch1a and Notch3 is required for maintaining zebrafish rhombomere boundaries

In vertebrates, hindbrain is subdivided into seven segments termed rhombomeres and the interface between each rhombomere forms the boundary. Similar to the D/V boundary formation in Drosophila, Notch activation has been shown to regulate the segregation of rhombomere boundary cells. Here we further explored the function of Notch signaling in the formation of rhombomere boundaries. By using bodipy ceramide cell-labeling technique, we found that the hindbrain boundary is formed initially in mib mutants but lost after 24 hours post-fertilization (hpf). This phenotype was more severe in mib ^ta52b allele than in mib ^tfi91 allele. Similarly, injection of su(h)-MO led to boundary defects in a dosage-dependent manner. Boundary cells were recovered in mib ^ta52b mutants in the hdac1-deficient background, where neurogenesis is inhibited. Furthermore, boundary cells lost sensitivity to reduced Notch activation from 15 somite stage onwards. We also showed that knockdown of notch3 function in notch1a mutants leads to the loss of rhombomere boundary cells and causes neuronal hyperplasia, indicating that Notch1a and Notch3 play a redundant role in the maintenance of rhombomere boundary.     

Distinctive properties of the interactions between Notch and Notch ligands

Although Notch signaling is known to be critical for the specification of cell fate in various developing organs, the particular roles of each Notch and Notch ligand (NotchL) have not yet been elucidated. The phenotypes found in loss-of-function experiments have varied, depending on the expression profiles of the receptors and ligands. However, in some cases, their significances differ from others, even with comparable levels of expression, suggesting a distinctive functional receptor-ligand interaction during the activation process of Notch signaling. In this review, the phenotypes observed in Notch/NotchL-deficient situations are introduced, and their distinct roles are accentuated. The distinctive features of the specific combinations of Notch/NotchL are also discussed. This review aims to highlight the unanswered questions in this field to help improve our understanding of the preferential functional interaction between Notch and NotchL.     

Notch signaling genes

Notch intercellular signaling is critical for diverse developmental pathways and for homeostasis in various types of stem cells and progenitor cells. Because Notch gene products need to be precisely regulated spatially and temporally, epigenetics is likely to help control expression of Notch signaling genes. Reduced representation bisulfite sequencing (RRBS) indicated significant hypomethylation in myoblasts, myotubes, and skeletal muscle vs. many nonmuscle samples at intragenic or intergenic regions of the following Notch receptor or ligand genes: NOTCH1, NOTCH2, JAG2, and DLL1. An enzymatic assay of sites in or near these genes revealed unusually high enrichment of 5-hydroxymethylcytosine (up to 81%) in skeletal muscle, heart, and cerebellum. Epigenetics studies and gene expression profiles suggest that hypomethylation and/or hydroxymethylation help control expression of these genes in heart, brain, myoblasts, myotubes, and within skeletal muscle myofibers. Such regulation could promote cell renewal, cell maintenance, homeostasis, and a poised state for repair of tissue damage.     

YTHDF2 Suppresses Notch Signaling through Post-transcriptional Regulation on Notch1

YTH domain family 2 (YTHDF2) is an N6-methyladenosine (m⁶A) binding protein promoting mRNA degradation in various biological processes. Despite its essential roles, the role of YTHDF2 in determining cell fates has not been fully elucidated. Notch signaling plays a vital role in determining cell fates, such as proliferation, differentiation, and apoptosis. We investigated the effect of YTHDF2 on Notch signaling. Our results show that YTHDF2 inhibits Notch signaling by downregulating the Notch1, HES1, and HES5 mRNA levels. Analyzing YTHDF2 deletion mutants indicates that the YTH domain is critical in regulating the Notch signal by directly binding m⁶A of Notch1 mRNA. Recently, YTHDF2 nuclear translocation was reported under heat shock conditions, but its physiological function is unknown. In our study, the YTH domain is required for YTHDF2 nuclear translocation. In addition, under heat shock stress, the Notch signal was significantly restored due to the increased expression of the Notch1 targets. These results suggest that YTHDF2 in the cytoplasm may act as an intrinsic suppressor in Notch signaling by promoting Notch1 mRNA degradation under normal cellular conditions. Conversely, upon the extracellular stress such as heat shock, YTHDF2 nuclear translocation resulting in reduced Notch1 mRNA decay may contribute to the increasing of Notch intracellular domain (NICD) regulating the survival-related target genes.     

The Notch regulator Numb links the Notch and TCR signaling pathways

Both the Notch and TCR signaling pathways play an important role in T cell development, but the links between these signaling pathways are largely unexplored. The adapter protein Numb is a well-characterized inhibitor of Notch and also contains a phosphotyrosine binding domain, suggesting that Numb could provide a link between these pathways. We explored this possibility by investigating the physical interactions among Notch, Numb, and the TCR signaling apparatus and by examining the consequences of a Numb mutation on T cell development. We found that Notch and Numb cocluster with the TCR at the APC contact during Ag-driven T cell-APC interactions in both immature and mature T cells. Furthermore, Numb coimmunoprecipitates with components of the TCR signaling apparatus. Despite this association, T cell development and T cell activation occur normally in the absence of Numb, perhaps due to the expression of the related protein, Numblike. Together our data suggest that Notch and TCR signals may be integrated at the cell membrane, and that Numb may be an important adapter in this process.