Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors.

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are essential for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. Although the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non-cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative-feedback loop with FGF signalling.

Desensitisation of Notch signalling through dynamic adaptation in the nucleus.

During embryonic development, signalling pathways orchestrate organogenesis by controlling tissue-specific gene expression programmes and differentiation. Although the molecular components of many common developmental signalling systems are known, our current understanding of how signalling inputs are translated into gene expression outputs in real-time is limited. Here we employ optogenetics to control the activation of Notch signalling during Drosophila embryogenesis with minute accuracy and follow target gene expression by quantitative live imaging. Light-induced nuclear translocation of the Notch Intracellular Domain (NICD) causes a rapid activation of target mRNA expression. However, target gene transcription gradually decays over time despite continuous photo-activation and nuclear NICD accumulation, indicating dynamic adaptation to the signalling input. Using mathematical modelling and molecular perturbations, we show that this adaptive transcriptional response fits to known motifs capable of generating near-perfect adaptation and can be best explained by state-dependent inactivation at the target cis-regulatory region. Taken together, our results reveal dynamic nuclear adaptation as a novel mechanism controlling Notch signalling output during tissue differentiation.

Exploring the therapeutic potential of plasma from intermittent fasting and untreated rats on aging-induced liver damage.

This research aims to investigate the effects of plasma from 12-month-old intermittently fasting rats (IFpls) and untreated rats (Npls) on the liver biomolecules and histological changes in 24-month-old male Sprague-Dawley rats. Fasting rats underwent an 18-h daily fasting period and a 6-h feeding window for 35 days. The plasma was administered bi-daily, and blood samples were examined for specific liver biomolecules. Fourier transform infrared (FTIR) spectroscopy and linear discriminant analysis (LDA) was used to identify molecular profiles. Liver sections were stained for histopathological evaluation, and the expression levels of Notch signalling pathway components were assessed. Distinct molecular profiles were identified across liver biomolecules, lipids, proteins and nucleic acids with high accuracy. Notably, IFpls was found to protect against hepatic instability, microvesicular steatosis and liver fibrosis by decreasing lymphatic infiltration density and Notch pathway expression levels. Both treatments reduced protein oxidation and carbonylation, with Npls showing a pronounced decrease in protein oxidation. Furthermore, Npls increased protein conformation and glycogen/phosphate content, while IFpls increased glucose/protein content. Both IFpls and Npls induce substantial and unique alterations in liver biomolecules. IFpls offers a protective effect on various liver conditions, while Npls exhibits promising results in reducing protein oxidation and altering biomolecule content. These findings offer valuable insights for future research and potential therapeutic approaches.

GHITM regulates malignant phenotype and sensitivity to PD-1 blockade of renal cancer cells via Notch signalling.

Growth hormone inducible transmembrane protein (GHITM), one member of Bax inhibitory protein-like family, has been rarely studied, and the clinical importance and biological functions of GHITM in kidney renal clear cell carcinoma (KIRC) still remain unknown. In the present study, we found that GHITM was downregulated in KIRC. Aberrant GHITM downregulation related to clinicopathological feature and unfavourable prognosis of KIRC patients. GHITM overexpression inhibited KIRC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, GHITM overexpression could induce the downregulation of Notch1, which acts as an oncogene in KIRC. Overexpression of Notch1 effectively rescued the inhibitory effect induced by GHITM upregulation. More importantly, GHITM could regulate PD-L1 protein abundance and ectopic overexpression of GHITM enhanced the antitumour efficiency of PD-1 blockade in KIRC, which provided new insights into antitumour therapy. Furthermore, we also showed that YY1 could decrease GHITM level via binding to its promoter. Taken together, our study revealed that GHITM was a promising therapeutic target for KIRC, which could modulate malignant phenotype and sensitivity to PD-1 blockade of renal cancer cells via Notch signalling pathway.

Identification of a Notch transcriptomic signature for breast cancer.

BACKGROUND: Dysregulated Notch signalling contributes to breast cancer development and progression, but validated tools to measure the level of Notch signalling in breast cancer subtypes and in response to systemic therapy are largely lacking. A transcriptomic signature of Notch signalling would be warranted, for example to monitor the effects of future Notch-targeting therapies and to learn whether altered Notch signalling is an off-target effect of current breast cancer therapies. In this report, we have established such a classifier. METHODS: To generate the signature, we first identified Notch-regulated genes from six basal-like breast cancer cell lines subjected to elevated or reduced Notch signalling by culturing on immobilized Notch ligand Jagged1 or blockade of Notch by γ-secretase inhibitors, respectively. From this cadre of Notch-regulated genes, we developed candidate transcriptomic signatures that were trained on a breast cancer patient dataset (the TCGA-BRCA cohort) and a broader breast cancer cell line cohort and sought to validate in independent datasets. RESULTS: An optimal 20-gene transcriptomic signature was selected. We validated the signature on two independent patient datasets (METABRIC and Oslo2), and it showed an improved coherence score and tumour specificity compared with previously published signatures. Furthermore, the signature score was particularly high for basal-like breast cancer, indicating an enhanced level of Notch signalling in this subtype. The signature score was increased after neoadjuvant treatment in the PROMIX and BEAUTY patient cohorts, and a lower signature score generally correlated with better clinical outcome. CONCLUSIONS: The 20-gene transcriptional signature will be a valuable tool to evaluate the response of future Notch-targeting therapies for breast cancer, to learn about potential effects on Notch signalling from conventional breast cancer therapies and to better stratify patients for therapy considerations.

The Clathrin adaptor AP-1 and Stratum act in parallel pathways to control Notch activation in Drosophila sensory organ precursors cells.

Drosophila sensory organ precursors divide asymmetrically to generate pIIa/pIIb cells, the identity of which relies on activation of Notch at cytokinesis. Although Notch is present apically and basally relative to the midbody at the pIIa-pIIb interface, the basal pool of Notch is reported to be the main contributor for Notch activation in the pIIa cell. Intra-lineage signalling requires appropriate apico-basal targeting of Notch, its ligand Delta and its trafficking partner Sanpodo. We have previously reported that AP-1 and Stratum regulate the trafficking of Notch and Sanpodo from the trans-Golgi network to the basolateral membrane. Loss of AP-1 or Stratum caused mild Notch gain-of-function phenotypes. Here, we report that their concomitant loss results in a penetrant Notch gain-of-function phenotype, indicating that they control parallel pathways. Although unequal partitioning of cell fate determinants and cell polarity were unaffected, we observed increased amounts of signalling-competent Notch as well as Delta and Sanpodo at the apical pIIa-pIIb interface, at the expense of the basal pool of Notch. We propose that AP-1 and Stratum operate in parallel pathways to localize Notch and control where receptor activation takes place.

A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis.

According to the current consensus, murine neural stem cells (NSCs) apically contacting the lateral ventricle generate differentiated progenitors by rare asymmetric divisions or by relocating to the basal side of the ventricular-subventricular zone (V-SVZ). Both processes will ultimately lead to the generation of adult-born olfactory bulb (OB) interneurons. In contrast to this view, we here find that adult-born OB interneurons largely derive from an additional NSC-type resident in the basal V-SVZ. Despite being both capable of self-renewal and long-term quiescence, apical and basal NSCs differ in Nestin expression, primary cilia extension and frequency of cell division. The expression of Notch-related genes also differs between the two NSC groups, and Notch activation is greatest in apical NSCs. Apical downregulation of Notch-effector Hes1 decreases Notch activation while increasing proliferation across the niche and neurogenesis from apical NSCs. Underscoring their different roles in neurogenesis, lactation-dependent increase in neurogenesis is paralleled by extra activation of basal but not apical NSCs. Thus, basal NSCs support OB neurogenesis, whereas apical NSCs impart Notch-mediated lateral inhibition across the V-SVZ.

Temporal cell fate determination in the spinal cord is mediated by the duration of Notch signalling.

During neural development, progenitor cells generate different types of neurons in specific time windows. Despite the characterisation of many of the transcription factor networks involved in these differentiation events, the mechanism behind their temporal regulation is poorly understood. To address this question, we studied the temporal differentiation of the simple lateral floor plate (LFP) domain in the zebrafish spinal cord. LFP progenitors generate both early-born Kolmer-Agduhr" (KA") interneuron and late-born V3 interneuron populations. Analysis using a Notch signalling reporter demonstrates that these cell populations have distinct Notch signalling profiles. Not only do V3 progenitors receive higher total levels of Notch response, but they collect this response over a longer duration compared to KA" progenitors. To test whether the duration of Notch signalling determines the temporal cell fate specification, we combined a transgene that constitutively activates Notch signalling in the ventral spinal cord with a heat shock inducible Notch signalling terminator to switch off Notch response at any given time. Sustained Notch signalling results in expanded LFP progenitors while KA" and V3 interneurons fail to specify. Early termination of Notch signalling leads to exclusively KA" cell fate, despite the high level of Notch signalling, whereas late attenuation of Notch signalling drives only V3 cell fate. This suggests that the duration of Notch signalling, not simply the level, mediates cell fate specification. Interestingly, knockdown experiments reveal a role for the Notch ligand Jag2b in maintaining LFP progenitors and limiting their differentiation into KA" and V3 interneurons. Our results indicate that Notch signalling is required for neural progenitor maintenance while a specific attenuation timetable defines the fate of the postmitotic progeny.

Dissecting Hes-centred transcriptional networks in neural stem cell maintenance and tumorigenesis in Drosophila.

Neural stem cells divide during embryogenesis and juvenile life to generate the entire complement of neurons and glia in the nervous system of vertebrates and invertebrates. Studies of the mechanisms controlling the fine balance between neural stem cells and more differentiated progenitors have shown that, in every asymmetric cell division, progenitors send a Delta-Notch signal to their sibling stem cells. Here, we show that excessive activation of Notch or overexpression of its direct targets of the Hes family causes stem-cell hyperplasias in the Drosophila larval central nervous system, which can progress to malignant tumours after allografting to adult hosts. We combined transcriptomic data from these hyperplasias with chromatin occupancy data for Dpn, a Hes transcription factor, to identify genes regulated by Hes factors in this process. We show that the Notch/Hes axis represses a cohort of transcription factor genes. These are excluded from the stem cells and promote early differentiation steps, most likely by preventing the reversion of immature progenitors to a stem-cell fate. We describe the impact of two of these 'anti-stemness' factors, Zfh1 and Gcm, on Notch/Hes-triggered tumorigenesis.

Notch signalling regulates epibranchial placode patterning and segregation.

Epibranchial placodes are the geniculate, petrosal and nodose placodes that generate parts of cranial nerves VII, IX and X, respectively. How the three spatially separated placodes are derived from the common posterior placodal area is poorly understood. Here, we reveal that the broad posterior placode area is first patterned into a Vgll2+/Irx5+ rostral domain and a Sox2+/Fgf3+/Etv5+ caudal domain relative to the first pharyngeal cleft. This initial rostral and caudal patterning is then sequentially repeated along each pharyngeal cleft for each epibranchial placode. The caudal domains give rise to the neuronal and non-neuronal cells in the placode, whereas the rostral domains are previously unrecognized structures, serving as spacers between the final placodes. Notch signalling regulates the balance between the rostral and caudal domains: high levels of Notch signalling expand the caudal domain at the expense of the rostral domain, whereas loss of Notch signalling produces the converse phenotype. Collectively, these data unravel a new patterning principle for the early phases of epibranchial placode development and a role for Notch signalling in orchestrating epibranchial placode segregation and differentiation.

Overexpression of notch signaling in renin cells leads to a polycystic kidney phenotype.

Polycystic kidney disease (PKD) is an inherited disorder that results in large kidneys, numerous fluid-filled cysts, and ultimately end-stage kidney disease. PKD is either autosomal dominant caused by mutations in PKD1 or PKD2 genes or autosomal recessive caused by mutations in the PKHD1 or DZIP1L genes. While the genetic basis of PKD is known, the downstream molecular mechanisms and signaling pathways that lead to deregulation of proliferation, apoptosis, and differentiation are not completely understood. The Notch pathway plays critical roles during kidney development including directing differentiation of various progenitor cells, and aberrant Notch signaling results in gross alternations in cell fate. In the present study, we generated and studied transgenic mice that have overexpression of an intracellular fragment of mouse Notch1 ('NotchIC') in renin-expressing cells. Mice with overexpression of NotchIC in renin-expressing cells developed numerous fluid-filled cysts, enlarged kidneys, anemia, renal insufficiency, and early death. Cysts developed in both glomeruli and proximal tubules, had increased proliferation marks, and had increased levels of Myc. The present work implicates the Notch signaling pathway as a central player in PKD pathogenesis and suggests that the Notch-Myc axis may be an important target for therapeutic intervention.

Polycystic kidneys: interaction of notch and renin.

Polycystic kidney disease (PKD) is a developmental disorder, which either manifests in early childhood or later in life, depending on the genetic mutation one harbors. The mechanisms of cyst initiation are not well understood. Increasing literature is now suggesting that Notch signaling may play a critical role in PKD. Activation of Notch signaling is important during nephrogenesis and slows down after development. Deletion of various Notch molecules in the cap mesenchyme leads to formation of cysts and early death in mice. A new study by Belyea et al. has now found that cells of renin lineage may link Notch expression and cystic kidney disease. Here, we use our understanding of Notch signaling and PKD to speculate about the significance of these interactions.

Basophil responses in susceptible AKR mice upon infection with the intestinal helminth parasite Trichuris muris.

Intestinal helminth infection promotes a Type 2 inflammatory response in resistant C57BL/6 mice that is essential for worm clearance. The study of inbred mouse strains has revealed factors that are critical for parasite resistance and delineated the role of Type 1 versus Type 2 immune responses in worm clearance. In C57BL/6 mice, basophils are key innate immune cells that promote Type 2 inflammation and are programmed via the Notch signalling pathway during infection with the helminth Trichuris muris. However, how the host genetic background influences basophil responses and basophil expression of Notch receptors remains unclear. Here we use genetically susceptible inbred AKR/J mice that have a Type 1-skewed immune response during T. muris infection to investigate basophil responses in a susceptible host. Basophil population expansion occurred in AKR/J mice even in the absence of fulminant Type 2 inflammation during T. muris infection. However, basophils in AKR/J mice did not robustly upregulate expression of the Notch2 receptor in response to infection as occurred in C57BL/6 mice. Blockade of the Type 1 cytokine interferon-γ in infected AKR/J mice was not sufficient to elicit infection-induced basophil expression of the Notch2 receptor. These data suggest that the host genetic background, outside of the Type 1 skew, is important in regulating basophil responses during T. muris infection in susceptible AKR/J mice.

Expression of Epsin3 and its interaction with Notch signalling in oral epithelial dysplasia and oral squamous cell carcinoma.

BACKGROUND: Most oral squamous cell carcinoma patients present with late-stage disease. Early detection of the disease is considered to be the most effective way of improving patient outcomes. Several biomarkers have been identified as indicators of oral cancer development and progression; however, none have been translated into clinical practice. In this study, we have investigated the role of Epsin3, an endocytic adaptor protein, and Notch1, a transmembrane signalling protein, in oral carcinogenesis with a view to explore their potential as biomarkers. METHODS: Oral cancer cell lines and a normal oral keratinocyte cell line were used together with tissue samples of normal oral mucosa (n = 21), oral epithelial dysplasia (n = 74) and early stage (Stages I and II) oral squamous cell carcinoma (n = 31). Immunocytochemical staining, immunoblotting and real-time quantitative polymerase chain reaction (PCR) were performed to assess protein as well as gene expression levels. RESULTS: The expression levels of Epsin3 and Notch1 mRNA and protein are variable across different oral squamous cell carcinoma derived cell lines. Epsin3 was upregulated in oral epithelial dysplasia and oral squamous cell carcinoma tissues compared with normal epithelium. Overexpression of Epsin3 resulted in a significant reduction of Notch1 expression in oral squamous cell carcinoma. Notch1 was generally downregulated in the dysplasia and oral squamous cell carcinoma samples. CONCLUSION: Epsin3 is upregulated in oral epithelial dysplasia and oral squamous cell carcinoma and has the potential to be used as a biomarker for oral epithelial dysplasia. Notch signalling is downregulated in oral squamous cell carcinoma, possibly through an Epsin3-induced de-activation pathway.

Autoregulation of Transcription and Translation: A Qualitative Analysis.

The regulation of both mRNA transcription and translation by down-stream gene products allows for a range of rich dynamical behaviours (e.g. homeostatic, oscillatory, excitability and intermittent solutions). Here, qualitative analysis is applied to an existing model of a gene regulatory network in which a protein dimer inhibits its own transcription and upregulates its own translation rate. It is demonstrated that the model possesses a unique steady state, conditions are derived under which limit cycle solutions arise and estimates are provided for the oscillator period in the limiting case of a relaxation oscillator. The analysis demonstrates that oscillations can arise only if mRNA is more stable than protein and the effect of nonlinear translation inhibition is sufficiently strong. Moreover, it is shown that the oscillation period can vary non-monotonically with transcription rate. Thus the proposed framework can provide an explanation for observed species-specific dependency of segmentation clock period on Notch signalling activity. Finally, this study facilitates the application of the proposed model to more general biological settings where post transcriptional regulation effects are likely important.

Notch activation promotes bone metastasis via SPARC inhibition in adenoid cystic carcinoma.

OBJECTIVES: We aimed to investigate bone metastasis induced by Notch signalling pathway dysregulation and to demonstrate that SPARC is a potential therapeutic target in adenoid cystic carcinoma (AdCC) with Notch dysregulation. MATERIALS AND METHODS: This retrospective study enrolled 144 AdCC patients. RNA-sequencing and enrichment analyses were performed using 32 AdCC samples. Osteonectin/SPARC and the Notch activation indicator Notch intracellular domain (NICD) were detected using immunohistochemistry. Cell proliferation and migration assays were conducted using stably NICD over-expressing cells. The effect of SPARC on osteoclast differentiation in NICD cells was investigated using western blotting, quantitative reverse transcription PCR, tartrate-resistant acid phosphatase staining and resorption assays. RESULTS: RNA-sequencing analysis showed that genes down-regulated in Notch-mutant AdCCs, such as SPARC, were enriched in ossification and osteoblast differentiation. Most (75/110, 68.2%) Notch1-wild-type AdCCs showed SPARC over-expression, whereas 30 out of 34 (88.2%) Notch1-mutant tumours showed low SPARC expression. SPARC over-expression was then found negatively to be correlated with NICD expression in 144 AdCCs. NICD over-expression promoted cell growth, migration and osteoclast differentiation, which could be partly reversed by exogenous SPARC. CONCLUSIONS: Notch activation in AdCC contributes to bone metastasis through SPARC inhibition. The study results suggest that SPARC may represent a prognostic biomarker and potential therapeutic target.

Ubiquitylation is required for the incorporation of the Notch receptor into intraluminal vesicles to prevent prolonged and ligand-independent activation of the pathway.

BACKGROUND: Ubiquitylation of the ligands and the receptor plays an important part in the regulation of the activity of the evolutionary conserved Notch signalling pathway. However, its function for activation of Notch is not completely understood, despite the identification of several E3 ligases devoted to the receptor. RESULTS: Here we analysed a variant of the Notch receptor where all lysines in its intracellular domain are replaced by arginines. Our analysis of this variant revealed that ubiquitylation of Notch is not essential for its endocytosis. We identified two functions for ubiquitylation of lysines in the Notch receptor. First, it is required for the degradation of free Notch intracellular domain (NICD) in the nucleus, which prevents a prolonged activation of the pathway. More importantly, it is also required for the incorporation of Notch into intraluminal vesicles of maturing endosomes to prevent ligand-independent activation of the pathway from late endosomal compartments. CONCLUSIONS: The findings clarify the role of lysine-dependent ubiquitylation of the Notch receptor and indicate that Notch is endocytosed by several independent operating mechanisms.

DLL3 expression and methylation are associated with lower-grade glioma immune microenvironment and prognosis.

Notch signalling pathway, particularly its ligand delta-ligand 3 (DLL3), is important in glioma, however, little is known about DLL3 regulation and prognostic effects. Immunohistochemistry on a cohort of 163 gliomas revealed DLL3 upregulation in IDH1 mutant gliomas, where it was associated with a favourable prognosis (HR[95% CI]: 0.28[0.09-0.87]; p = 0.021). We investigated the epigenetic regulation of DLL3, and identified individual CpG sites correlating with DLL3 mRNA expression, which were significant prognostic markers in LGG. In silico analysis revealed that infiltrating immune cells significantly correlated with DLL3 expression, methylation and somatic copy number alterations. The prognostic effects of DLL3 expression was significantly affected by infiltration of immune cells. RNA Sequencing of 83 LGGs and GO Term analysis of differentially expressed genes showed that low DLL3 expression was related to ciliogenesis, which was confirmed by TCGA LGG analysis. Thus, DLL3 may play an important role in the immune microenvironment and prognosis of LGGs.

Regulation of Notch1 Signalling by Long Non-Coding RNAs in Cancers and Other Health Disorders.

Notch1 signalling plays a multifaceted role in tissue development and homeostasis. Currently, due to the pivotal role of Notch1 signalling, the relationship between NOTCH1 expression and the development of health disorders is being intensively studied. Nevertheless, Notch1 signalling is not only controlled at the transcriptional level but also by a variety of post-translational events. First is the ligand-dependent mechanical activation of NOTCH receptors and then the intracellular crosstalk with other signalling molecules-among those are long non-coding RNAs (lncRNAs). In this review, we provide a detailed overview of the specific role of lncRNAs in the modulation of Notch1 signalling, from expression to activity, and their connection with the development of health disorders, especially cancers.

A Review of the Molecular Landscape of Adenoid Cystic Carcinoma of the Lacrimal Gland.

Adenoid cystic carcinoma (ACC) has a worldwide incidence of three to four cases per million population. Although more cases occur in the minor and major salivary glands, it is the most common lacrimal gland malignancy. ACC has a low-grade, indolent histological appearance, but is relentlessly progressive over time and has a strong proclivity to recur and/or metastasise. Current treatment options are limited to complete surgical excision and adjuvant radiotherapy. Intra-arterial systemic therapy is a recent innovation. Recurrent/metastatic disease is common due to perineural invasion, and it is largely untreatable as it is refractory to conventional chemotherapeutic agents. Given the rarity of this tumour, the molecular mechanisms that govern disease pathogenesis are poorly understood. There is an unmet, critical need to develop effective, personalised targeted therapies for the treatment of ACC in order to reduce morbidity and mortality associated with the disease. This review details the evidence relating to the molecular underpinnings of ACC of the lacrimal gland, including the MYB-NFIB chromosomal translocations, Notch-signalling pathway aberrations, DNA damage repair gene mutations and epigenetic modifications.