Temperature-Sensitive Mutations of the Notch Locus in DROSOPHILA MELANOGASTER

Temperature-conditional mutations of the Notch locus were characterized in an attempt to understand the organization of a "complex locus" and the control of its function in development. Among 21 newly induced Notch alleles, about one-half are temperature-conditional for some effects, and three are temperature-sensitive for viability. One temperature-sensitive lethal, l(1)N^ts1, is functionally non-complementing for all known effects of Notch locus mutations and maps at a single site within the locus. Among the existing alleles involved in complex patterns of interallelic complementation, Ax^59d5 is found to be temperature-sensitive, while fa ^g, spl, and l(1)N are temperature-independent. Whereas temperature-sensitive alleles map predominantly to the right-most fifth of the locus, fa^g, spl, and l(1)N are known to map to the left of this region. Temperature-shift experiments demonstrate that fa^g, spl, and l(1)N cause defects at specific, non-overlapping times in development.—We conclude (1) that the Notch locus is a single cistron (responsible for a single functional molecule, presumably a polypeptide); (2) that the right-most fifth of the locus is, at least in part, the region involved in coding for the Notch product; (3) that the complexity of interallelic complementation is a developmental effect of mutations that cause defects at selected times and spaces, and that complementation occurs because the mutant defects are temporally and spatially non-overlapping; and (4) that mutants express selected defects due to critical temporal and spatial differences in the chemical conditions controlling the synthesis or function of the Notch product. The complexity of the locus appears to reside in controlling the expression (synthesis or function) of the Notch product in development.     

The Different Role of Notch1 and Notch2 in Astrocytic Gliomas

It is well known that Notch signaling plays either oncogenic or tumor suppressive role in a variety of tumors, depending on the cellular context. However, in our previous study, we found that Notch1 was overexpressed while Notch2 downregulated in the majority of astrocytic gliomas with different grades as well as in glioblastoma cell lines U251 and A172. We had knocked down Notch1 by siRNA in glioblastoma cells, and identified that the cell growth and invasion were inhibited, whereas cell apoptosis was induced either in vitro or in vivo. For further clarification of the role of Notch2 in pathogenesis of gliomas, enforced overexpression of Notch2 was carried out with transfection of Notch2 expression plasmid in glioma cells and the cell growth, invasion and apoptosis were examined in vitro and in vivo in the present study, and siRNA targeting Notch1 was used as a positive control in vivo. The results showed that upregulating Notch2 had the effect of suppressing cell growth and invasion as well as inducing apoptosis, just the same as the results of knocking down Notch1. Meanwhile, the activity of core signaling pathway–EGFR/PI3K/AKT in astrocytic glioma cells was repressed. Thus, the present study reveals, for the first time, that Notch1 and Notch2 play different roles in the biological processes of astrocytic gliomas. Knocking down the Notch1 or enforced overexpression of Notch2 both modulate the astrocytic glioma phenotype, and the mechanism by which Notch1 and 2 play different roles in the glioma growth should be further investigated.     

Spatiotemporal expression of Notch receptors and ligands in developing mouse placenta

Notch signaling is involved in cell lineage specification in many developing organs. In mice there are four known Notch receptor genes (Notch1–4) and five ligands genes (Dll1, 3, 4 and Jagged1 and 2). Notch2 is essential for development of placenta, an organ that mediates feto-maternal nutrient and gas exchange as well as maternal adaptations to pregnancy. However the role of other Notch receptors and ligands in placentation is not known. In order to gain better insight into the role of Notch signaling in mouse placenta we thoroughly analyzed mRNA expression of all Notch receptors and ligands in all trophoblast cell types from the embryonic day (E) 7.5 to E12.5, the period during which all of the substructures of the placenta develop. Here we show that Notch receptors and ligands are specifically and dynamically expressed in multiple cell layers of developing placenta. We found that the Notch2 receptor and Jagged1 and Jagged2 ligand genes are complementarily expressed in trophoblast cells of the chorion and its later derivatives in the labyrinth. Dll4 and Notch2 expression complement each other in the ectoplacental cone, while Dll1 and Notch2 are expressed in an ectoplacental cone derivative, the junctional zone. Moreover Dll4 and Notch2 are expressed at the ectoplacental cone–decidua interface at early stages of placentation. Additionally we show that Notch2 is dynamically expressed in all trophoblast giant cell subtypes, which is consistent with previous reports. Overall these expression pattern results suggest that Notch signaling may play several diverse roles during placenta development.     

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.     

Negative Complementation at the Notch Locus of DROSOPHILA MELANOGASTER

Four Abruptex alleles (Ax^E1, Ax^E₂, Ax^₉B₂, and Ax¹⁶¹⁷²) have been mapped within the Notch locus. Based on their visible phenotypes and their interactions with one another and with N mutations, the Ax alleles can be divided into two groups. Heterozygous combinations of members of the same group are intermediate in phenotype compared to the respective homozygotes, whereas heterozygotes of Ax alleles from different groups exhibit negative heterosis, being much less viable and more extremely mutant than either homozygote. It is suggested that the Notch locus is a multi-functional regulator ("integrator") gene, whose product possesses both "repressor" and "activator" functions for the processes it regulates.     

Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors

Background

Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.

Principal Findings

Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC₅₀ values as low as 5±3 nM and 0.13±0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR “class I” point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare “class II” or “class III” mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.

Conclusions/Significance

Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.     

The contribution of Notch1 to nephron segmentation in the developing kidney is revealed in a sensitized Notch2 background and can be augmented by reducing Mint dosage

We previously determined that Notch2, and not Notch1, was required for forming proximal nephron segments. The dominance of Notch2 may be conserved in humans, since Notch2 mutations occur in Alagille syndrome (ALGS) 2 patients, which includes renal complications. To test whether mutations in Notch1 could increase the severity of renal complications in ALGS, we inactivated conditional Notch1 and Notch2 alleles in mice using a Six2-GFP::Cre. This BAC transgene is expressed mosaically in renal epithelial progenitors but uniformly in cells exiting the progenitor pool to undergo mesenchymal-to-epithelial transition. Although delaying Notch2 inactivation had a marginal effect on nephron numbers, it created a sensitized background in which the inactivation of Notch1 severely compromised nephron formation, function, and survival. These and additional observations indicate that Notch1 in concert with Notch2 contributes to the morphogenesis of renal vesicles into S-shaped bodies in a RBP-J-dependent manner. A significant implication is that elevating Notch1 activity could improve renal functions in ALGS2 patients. As proof of principle, we determined that conditional inactivation of Mint, an inhibitor of Notch-RBP-J interaction, resulted in a moderate rescue of Notch2 null kidneys, implying that temporal blockage of Notch signaling inhibitors downstream of receptor activation may have therapeutic benefits for ALGS patients.     

Hesr1 and Hesr2 may act as early effectors of Notch signaling in the developing cochlea

In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development.     

Negative Regulation of Notch Signaling by Xylose

The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14–20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16–20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16–20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16–20 of Notch.     

Notch Signaling in Osteocytes Differentially Regulates Cancellous and Cortical Bone Remodeling

Notch receptors play a role in skeletal development and homeostasis, and Notch activation in undifferentiated and mature osteoblasts causes osteopenia. In contrast, Notch activation in osteocytes increases bone mass, but the mechanisms involved and exact functions of Notch are not known. In this study, Notch1 and -2 were inactivated preferentially in osteocytes by mating Notch1/2 conditional mice, where Notch alleles are flanked by loxP sequences, with transgenics expressing Cre directed by the Dmp1 (dentin matrix protein 1) promoter. Notch1/2 conditional null male and female mice exhibited an increase in trabecular bone volume due to an increase in osteoblasts and decrease in osteoclasts. In male null mice, this was followed by an increase in osteoclast number and normalization of bone volume. To activate Notch preferentially in osteocytes, Dmp1-Cre transgenics were crossed with Rosa^Notch mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and Notch1 intracellular domain sequences. Dmp1-Cre^+/−;Rosa^Notch mice exhibited an increase in trabecular bone volume due to decreased bone resorption and an increase in cortical bone due to increased bone formation. Biomechanical and chemical properties were not affected. Osteoprotegerin mRNA was increased, sclerostin and dickkopf1 mRNA were decreased, and Wnt signaling was enhanced in Dmp1-Cre^+/−;Rosa^Notch femurs. Botulinum toxin A-induced muscle paralysis caused pronounced osteopenia in control mice, but bone mass was preserved in mice harboring the Notch activation in osteocytes. In conclusion, Notch plays a unique role in osteocytes, up-regulates osteoprotegerin and Wnt signaling, and differentially regulates trabecular and cortical bone homeostasis.     

Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D)-induced skin carcinogenesis in vivo

Background

The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras^G12D mice with ablation of Notch1 and/or Notch2.

Methodology/Principal Findings

Surprisingly, mice with activated Kras^G12D and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.

Conclusions/Significance

Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.     

Significance of glycosylation in Notch signaling

Notch signaling is essential for cell-fate specification in metazoans, and dysregulation of the pathway leads to a variety of human diseases including heart and vascular defects as well as cancer. Glycosylation of the Notch extracellular domain has emerged as an elegant means for regulating Notch activity, especially since the discovery that Fringe is a glycosyltransferase that modifies O-fucose in 2000. Since then, several other O-glycans on the extracellular domain have been demonstrated to modulate Notch activity. Here we will describe recent results on the molecular mechanisms by which Fringe modulates Notch activity, summarize recent work on how O-glucose, O-GlcNAc, and O-GalNAc glycans affect Notch, and discuss several human genetic disorders resulting from defects in Notch glycosylation.     

Making sense out of missense mutations: Mechanistic dissection of Notch receptors through structure-function studies in Drosophila

Notch signaling is involved in the development of almost all organ systems and is required post-developmentally to modulate tissue homeostasis. Rare variants in Notch signaling pathway genes are found in patients with rare Mendelian disorders, while unique or recurrent somatic mutations in a similar set of genes are identified in cancer. The human genome contains four genes that encode Notch receptors, NOTCH1-4, all of which are linked to genetic diseases and cancer. Although some mutations have been classified as clear loss- or gain-of-function alleles based on cellular or rodent based assay systems, the functional consequence of many variants/mutations in human Notch receptors remain unknown. In this review, I will first provide an overview of the domain structure of Notch receptors and discuss how each module is known to regulate Notch signaling activity in vivo using the Drosophila Notch receptor as an example. Next, I will introduce some interesting mutant alleles that have been isolated in the fly Notch gene over the past > 100 years of research and discuss how studies of these mutations have facilitated the understanding of Notch biology. By identifying unique alleles of the fly Notch gene through forward genetic screens, mapping their molecular lesions and characterizing their phenotypes in depth, one can begin to unravel new mechanistic insights into how different domains of Notch fine-tune signaling output. Such information can be useful in deciphering the functional consequences of rare variants/mutations in human Notch receptors, which in turn can influence disease management and therapy.     

Potential Role of Notch Signalling in CD34+ Chronic Myeloid Leukaemia Cells: Cross-Talk between Notch and BCR-ABL

Notch signalling is critical for haemopoietic stem cell (HSC) self-renewal and survival. The role of Notch signalling has been reported recently in chronic myeloid leukaemia (CML) – a stem cell disease characterized by BCR-ABL tyrosine kinase activation. Therefore, we studied the relationship between BCR-ABL and Notch signalling and assessed the expression patterns of Notch and its downstream target Hes1 in CD34⁺ stem and progenitor cells from chronic-phase CML patients and bone marrow (BM) from normal subjects (NBM). We found significant upregulation (p<0.05) of Notch1, Notch2 and Hes1 on the most primitive CD34⁺Thy⁺ subset of CML CD34⁺ cells suggesting that active Notch signalling in CML primitive progenitors. In addition, Notch1 was also expressed in distinct lymphoid and myeloid progenitors within the CD34⁺ population of primary CML cells. To further delineate the possible role and interactions of Notch with BCR-ABL in CD34⁺ primary cells from chronic-phase CML, we used P-crkl detection as a surrogate assay of BCR-ABL tyrosine kinase activity. Our data revealed that Imatinib (IM) induced BCR-ABL inhibition results in significant (p<0.05) upregulation of Notch activity, assessed by Hes1 expression. Similarly, inhibition of Notch leads to hyperactivation of BCR-ABL. This antagonistic relationship between Notch and BCR-ABL signalling was confirmed in K562 and ALL-SIL cell lines. In K562, we further validated this antagonistic relationship by inhibiting histone deacetylase (HDAC) - an effector pathway of Hes1, using valproic acid (VPA) - a HDAC inhibitor. Finally, we also confirmed the potential antagonism between Notch and BCR/ABL in In Vivo, using publically available GSE-database, by analysing gene expression profile of paired samples from chronic-phase CML patients pre- and post-Imatinib therapy. Thus, we have demonstrated an antagonistic relationship between Notch and BCR-ABL in CML. A combined inhibition of Notch and BCR-ABL may therefore provide superior clinical response over tyrosine-kinase inhibitor monotherapy by targeting both quiescent leukaemic stem cells and differentiated leukaemic cells and hence must be explored.     

Notch Pathway Is Activated by MAPK Signaling and Influences Papillary Thyroid Cancer Proliferation

Mutually exclusive genetic alterations in the RET, RAS, or BRAF genes, which result in constitutively active mitogen-activated protein kinase (MAPK) signaling, are present in about 70% of papillary thyroid carcinomas (PTCs). However, the effect of MAPK activation on other signaling pathways involved in oncogenic transformation, such as Notch, remains unclear. In this study, we tested the hypothesis that the MAPK pathway regulates Notch signaling and that Notch signaling plays a role in PTC cell proliferation. Conditional induction of MAPK signaling oncogenes RET/PTC3 or BRAF^T1799A in normal rat thyroid cell line mediated activation of Notch signaling, upregulating Notch1 receptor and Hes1, the downstream effector of Notch pathway. Conversely, pharmacological inhibition of MAPK reduced Notch signaling in PTC cell. Thyroid tumor samples from transgenic mice expressing BRAF^T1799A and primary human PTC samples showed high levels of Notch1 expression. Down-regulation of Notch signaling by γ-secretase inhibitor (GSI) or NOTCH1 RNA interference reduces PTC cell proliferation. Moreover, the combination of GSI with a MAPK inhibitor enhanced the growth suppression in PTC cells. This study revealed that RET/PTC and BRAF^T1799A activate Notch signaling and promote tumor growth in thyroid follicular cell. Taken together, these data suggest that Notch signaling may be explored as an adjuvant therapy for thyroid papillary cancer.     

Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7⁺ and Myf5⁻) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.KEY WORDS: Muscular dystrophy, Notch signaling, Stem cell