Notch Signaling Components Research Articles

Numb Suppresses Notch-Dependent Activation of Enhancer of split during Lateral Inhibition in the Drosophila Embryonic Nervous System.

The role of Drosophila numb in regulating Notch signaling and neurogenesis has been extensively studied, with a particular focus on its effects on the peripheral nervous system (PNS). Previous studies based on a single loss-of-function allele of numb, numb1, showed an antineurogenic effect on the peripheral nervous system (PNS), which revealed that the wild-type numb suppresses Notch signaling. In the current study, we examined whether this phenotype is consistently observed in loss-of-function mutations of numb. Two more numb alleles, numbEY03840 and numbEY03852, were shown to have an antineurogenic phenotype in the PNS. We also found that introducing a wild-type numb genomic fragment into numb1 homozygotes rescued their antineurogenic phenotype. These results demonstrated that loss-of-function mutations of numb universally induce this phenotype. Many components of Notch signaling are encoded by maternal effect genes, but no maternal effect of numb was observed in this study. The antineurogenic phenotype of numb was found to be dependent on the Enhancer of split (E(spl)), a downstream gene of Notch signaling. We found that the combination of E(spl) homozygous and numb1 homozygous suppressed the neurogenic phenotype of the embryonic central nervous system (CNS) associated with the E(spl) mutation. In the E(spl) allele, genes encoding basic helix-loop-helix proteins, such as m5, m6, m7, and m8, remain. Thus, in the E(spl) allele, derepression of Notch activity by numb mutation can rescue the neurogenic phenotype by increasing the expression of the remaining genes in the E(spl) complex. We also uncovered a role for numb in regulating neuronal projections. Our results further support an important role for numb in the suppression of Notch signaling during embryonic nervous system development.

Temporal Transcriptomic Profiling of the Developing Xenopus laevis Eye.

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development, including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and include regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth.

Temporal Transcriptomic Profiling of the Developing Xenopus laevis Eye.

Retinal progenitor cells (RPCs) are a multipotent and highly proliferative population that give rise to all retinal cell types during organogenesis. Defining their molecular signature is a key step towards identifying suitable approaches to treat visual impairments. Here, we performed RNA-sequencing of whole eyes from Xenopus at three embryonic stages and used differential expression analysis to define the transcriptomic profiles of optic tissues containing proliferating and differentiating RPCs during retinogenesis. Gene Ontology and KEGG pathway analyses showed that genes associated with developmental pathways (including Wnt and Hedgehog signaling) were upregulated during the period of active RPC proliferation in early retinal development (Nieuwkoop Faber st. 24 and 27). Developing eyes had dynamic expression profiles and shifted to enrichment for metabolic processes and phototransduction during RPC progeny specification and differentiation (st. 35). Furthermore, conserved adult eye regeneration genes were also expressed during early retinal development including sox2, pax6, nrl, and Notch signaling components. The eye transcriptomic profiles presented here span RPC proliferation to retinogenesis and included regrowth-competent stages. Thus, our dataset provides a rich resource to uncover molecular regulators of RPC activity and will allow future studies to address regulators of RPC proliferation during eye repair and regrowth.

Evolution and Function of the Notch Signaling Pathway: An Invertebrate Perspective.

The highly conserved Notch signaling pathway affects embryonic development, neurogenesis, homeostasis, tissue repair, immunity, and numerous other essential processes. Although previous studies have demonstrated the location and function of the core components of Notch signaling in various animal phyla, a more comprehensive summary of the Notch core components in lower organisms is still required. In this review, we objectively summarize the molecular features of the Notch signaling pathway constituents, their current expression profiles, and their functions in invertebrates, with emphasis on their effects on neurogenesis and regeneration. We also analyze the evolution and other facets of Notch signaling and hope that the contents of this review will be useful to interested researchers.

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.

Notch signaling as a master regulator of adult neurogenesis.

Neurogenesis ceases in most regions of the mammalian brain before or shortly after birth, however, in a few restricted brain regions, the production of new neurons proceeds into adulthood. Neural stem cells (NSCs) in these neurogenic zones are integrated into niches that control their activity and fate. Most stem cells in the adult brain are mitotically inactive and these cells can remain quiescent for months or even years. One of the key questions is what are the molecular mechanisms that regulate NSC maintenance and differentiation. Notch signaling has been shown to be a critical regulator of stem cell activity and maintenance in many tissues including in the nervous system. In this mini-review we discuss the roles of Notch signaling and the functions of the different Notch receptors and ligands in regulating neurogenesis in the adult murine brain. We review the functions of Notch signaling components in controlling NSC quiescence and entry into cell cycle and neurogenesis.

Mapping of Notch signaling in the developing organ of Corti in common marmosets.

The well-regulated development of the sensory epithelium is essential for hearing. This process involves the specification of a pro-sensory epithelium containing common progenitors that differentiate into hair and supporting cells. Notch signaling is one of the most critical pathways during these processes, and its modification is thought to be a feasible approach for treating hearing loss. Despite interspecies differences between rodents and primates or humans, most of our current knowledge regarding cochlear development has been obtained from rodent models. We therefore examined and mapped the expression patterns of Notch signal components in the developing cochlea of the common marmoset (Callithrix jacchus), a small monkey species native to the New World, a primate model animal. In contrast to the preserved expression patterns of the Notch signaling components in the hair cell differentiation between primates and rodents, we unveiled relatively large interspecies differences during the maturation of supporting cells. This improved knowledge of Notch signaling during primate cochlear development will facilitate the development of future regenerative therapies.

MYCN Amplification Is Associated with Reduced Expression of Genes Encoding γ-Secretase Complex and NOTCH Signaling Components in Neuroblastoma.

Amplification of the MYCN oncogene is found in ~20% of neuroblastoma (NB) cases and correlates with high-risk disease and poor prognosis. Despite the plethora of studies describing the role of MYCN in NB, the exact molecular mechanisms underlying MYCN's contribution to high-risk disease are not completely understood. Herein, we implemented an integrative approach combining publicly available RNA-Seq and MYCN ChIP-Seq datasets derived from human NB cell lines to define biological processes directly regulated by MYCN in NB. Our approach revealed that MYCN-amplified NB cell lines, when compared to non-MYCN-amplified cell lines, are characterized by reduced expression of genes involved in NOTCH receptor processing, axoneme assembly, and membrane protein proteolysis. More specifically, we found genes encoding members of the γ-secretase complex, which is known for its ability to liberate several intracellular signaling molecules from membrane-bound proteins such as NOTCH receptors, to be down-regulated in MYCN-amplified NB cell lines. Analysis of MYCN ChIP-Seq data revealed an enrichment of MYCN binding at the transcription start sites of genes encoding γ-secretase complex subunits. Notably, using publicly available gene expression data from NB primary tumors, we revealed that the expression of γ-secretase subunits encoding genes and other components of the NOTCH signaling pathway was also reduced in MYCN-amplified tumors and correlated with worse overall survival in NB patients. Genetic or pharmacological depletion of MYCN in NB cell lines induced the expression of γ-secretase genes and NOTCH-target genes. Chemical inhibition of γ-secretase activity dampened the expression of NOTCH-target genes upon MYCN depletion in NB cells. In conclusion, this study defines a set of MYCN-regulated pathways that are specific to MYCN-amplified NB tumors, and it suggests a novel role for MYCN in the suppression of genes of the γ-secretase complex, with an impact on the NOTCH-target gene expression in MYCN-amplified NB.

The mitochondrial ribosomal protein mRpL4 regulates Notch signaling.

Mitochondrial ribosomal proteins (MRPs) assemble as specialized ribosome to synthesize mtDNA-encoded proteins, which are essential for mitochondrial bioenergetic and metabolic processes. MRPs are required for fundamental cellular activities during animal development, but their roles beyond mitochondrial protein translation are poorly understood. Here, we report a conserved role of the mitochondrial ribosomal protein L4 (mRpL4) in Notch signaling. Genetic analyses demonstrate that mRpL4 is required in the Notch signal-receiving cells to permit target gene transcription during Drosophila wing development. We find that mRpL4 physically and genetically interacts with the WD40 repeat protein wap and activates the transcription of Notch signaling targets. We show that human mRpL4 is capable of replacing fly mRpL4 during wing development. Furthermore, knockout of mRpL4 in zebrafish leads to downregulated expression of Notch signaling components. Thus, we have discovered a previously unknown function of mRpL4 during animal development.

Integration of deep learning-based histopathology and transcriptomics reveals key genes associated with fibrogenesis in patients with advanced NASH

Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease globally and a leading cause for liver transplantation in the US. Its pathogenesis remains imprecisely defined. We combined two high-resolution modalities to tissue samples from NASH clinical trials, machine learning (ML)-based quantification of histological features and transcriptomics, to identify genes that are associated with disease progression and clinical events. A histopathology-driven 5-gene expression signature predicted disease progression and clinical events in patients with NASH with F3 (pre-cirrhotic) and F4 (cirrhotic) fibrosis. Notably, the Notch signaling pathway and genes implicated in liver-related diseases were enriched in this expression signature. In a validation cohort where pharmacologic intervention improved disease histology, multiple Notch signaling components were suppressed.

Carbon Ion Irradiation Downregulates Notch Signaling in Glioma Cell Lines, Impacting Cell Migration and Spheroid Formation.

Photon-based radiotherapy upregulates Notch signaling in cancer, leading to the acquisition of the stem cell phenotype and induction of invasion/migration, which contributes to the development of resistance to therapy. However, the effect of carbon ion radiotherapy (CIRT) on Notch signaling in glioma and its impact on stemness and migration is not explored yet. Human glioma cell lines (LN229 and U251), stable Notch1 intracellular domain (N1ICD) overexpressing phenotype of LN229 cells, and Notch inhibitor resistant LN229 cells (LN229R) were irradiated with either photon (X-rays) or (carbon ion irradiation) CII, and expressions of Notch signaling components were accessed by RT-PCR, Western blotting, and enzymatic assays and flow cytometry. Spheroid forming ability, cell migration, and clonogenic assay were used to evaluate the effect of modulated Notch signaling by irradiation. Our results show that X-ray irradiation induced the expression of Notch signaling components such as Notch receptors, target genes, and ADAM17 activity, while CII reduced it in glioma cell lines. The differential modulation of ADAM17 activity by CII and X-rays affected the cell surface levels of NOTCH1 and NOTCH2 receptors, as they were reduced by X-ray irradiation but increased in response to CII. Functionally, CII reduced the spheroid formation and migration of glioma cells, possibly by downregulating the N1ICD, as stable overexpression of N1ICD rescued these inhibitory effects of CII. Moreover, LN229R that are less reliant on Notch signaling for their survival showed less response to CII. Therefore, downregulation of Notch signaling resulting in the suppression of stemness and impaired cell migration by CII seen here may reduce tumor regrowth and disease dissemination, in addition to the well-established cytotoxic effects.

A comprehensive role evaluation and mechanism exploration of POGLUT2 in pan-cancer.

ObjectiveTo evaluate the role of POGLUT2 in pan-cancer through bioinformatics analysis and experimental verification.MethodsExpression, gene mutation and amplification, methylation, and copy number alteration (CNA) of POGLUT2 were evaluated using The Cancer Genome Atlas (TCGA), Cancer Cell Line Encyclopedia (CCLE), and Genotype-Tissue Expression (GTEx) databases. Moreover, POGLUT2 on survival and disease progression in pan-cancer was performed using TCGA data. Immune infiltration and tumor microenvironment evaluations were assessed by ImmuneScore, ImmuCellAI, and TIMER databases. POGLUT2 correlated drug resistance analysis was performed using the GDSC2 database. Furthermore, POGLUT2 knockdown of breast cancer cells was established, followed by in vitro biological function assays and in vivo tumor growth study. The mechanisms of POGLUT2 in breast cancer were briefly evaluated via its connection with Notch signaling.ResultsIncreased levels of POGLUT2 were found in multiple types of cancer tissues and cell lines. Moreover, increased gene mutation and amplification, methylation, and CNA of POGLUT2 were found in several types of cancers. POGLUT2 was mainly expressed in stromal cells as verified by StromalScore, ESTIMATEScore, ImmuneScore, and Tumor purity, and POGLUT2 was positively correlated with cancer-associated fibroblasts, macrophages, monocytes, and neutrophils in the tumor microenvironment. In vitro and in vivo results showed that POGLUT2 knockdown could delay tumor growth and progression. Notch signaling components were related to the function of POGLUT2.ConclusionsIncreased levels of POGLUT2 could result in the dysregulated immune cell infiltration and tumor microenvironment and showed a significant regulatory effect on the progression of breast cancer through Notch-related signaling.

RNA m6A demethylase ALKBH5 regulates the development of γδ T cells

γδ T cells are an abundant T cell population at the mucosa and are important in providing immune surveillance as well as maintaining tissue homeostasis. However, despite γδ T cells' origin in the thymus, detailed mechanisms regulating γδ T cell development remain poorly understood. N6-methyladenosine (m6A) represents one of the most common posttranscriptional modifications of messenger RNA (mRNA) in mammalian cells, but whether it plays a role in γδ T cell biology is still unclear. Here, we show that depletion of the m6A demethylase ALKBH5 in lymphocytes specifically induces an expansion of γδ T cells, which confers enhanced protection against gastrointestinal Salmonella typhimurium infection. Mechanistically, loss of ALKBH5 favors the development of γδ T cell precursors by increasing the abundance of m6A RNA modification in thymocytes, which further reduces the expression of several target genes including Notch signaling components Jagged1 and Notch2. As a result, impairment of Jagged1/Notch2 signaling contributes to enhanced proliferation and differentiation of γδ T cell precursors, leading to an expanded mature γδ T cell repertoire. Taken together, our results indicate a checkpoint role of ALKBH5 and m6A modification in the regulation of γδ T cell early development.

Sox11b regulates the migration and fate determination of Müller glia-derived progenitors during retina regeneration in zebrafish.

The transcription factor Sox11 plays important roles in retinal neurogenesis during vertebrate eye development. However, its function in retina regeneration remains elusive. Here we report that Sox11b, a zebrafish Sox11 homolog, regulates the migration and fate determination of Müller glia-derived progenitors (MGPCs) in an adult zebrafish model of mechanical retinal injury. Following a stab injury, the expression of Sox11b was induced in proliferating MGPCs in the retina. Sox11b knockdown did not affect MGPC formation at 4 days post-injury, although the nuclear morphology and subsequent radial migration of MGPCs were altered. At 7 days post-injury, Sox11b knockdown resulted in an increased proportion of MGPCs in the inner retina and a decreased proportion of MGPCs in the outer nuclear layer, compared with controls. Furthermore, Sox11b knockdown led to reduced photoreceptor regeneration, while it increased the numbers of newborn amacrines and retinal ganglion cells. Finally, quantitative polymerase chain reaction analysis revealed that Sox11b regulated the expression of Notch signaling components in the retina, and Notch inhibition partially recapitulated the Sox11b knockdown phenotype, indicating that Notch signaling functions downstream of Sox11b. Our findings imply that Sox11b plays key roles in MGPC migration and fate determination during retina regeneration in zebrafish, which may have critical implications for future explorations of retinal repair in mammals.

Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in oesophageal adenocarcinoma

ObjectiveOesophageal adenocarcinoma (EAC) arises in the setting of Barrett’s oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic...

Abstract 6071: Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in esophageal adenocarcinoma

Abstract Esophageal adenocarcinoma (EAC) is one of the most lethal of all human malignancies. EAC arises in the setting of Barrett’s esophagus, which is an intestinal metaplastic precursor lesion that develops from chronic reflux of gastrointestinal contents (especially acidic bile salts). Here we report that NOTCH signaling is activated in the esophagus through a unique pathway during exposure to acidic bile salts and progression to EAC. Using a combination of public databases, EAC cell line models, transgenic mice, and patient tissue samples, we detected significant upregulation of several NOTCH signaling components in EAC. Activated NOTCH signaling was confirmed by nuclear accumulation of NOTCH1 cleaved fragment (NICD) with corresponding up-regulation of NOTCH targets in EAC cells in response to acidic bile salts. Moreover, we identified DLL1 as the predominant ligand contributing NOTCH1 activation. Remarkably, DLL1 was regulated by direct cross talk between redox and inflammatory pathways that are activated during both reflux and malignant transformation. Mechanistically, the APE1 redox function transcriptionally up-regulated NF-κB in response to bile salts. This licensed NF-κB to transcriptionally up-regulate DLL1 to activate and stimulate downstream NOTCH1 signaling, thereby defining a novel APE1-NF-kB-NOTCH pro-tumorigenic pathway. This pathway was important for maintaining tumor initiating (cancer “stem cell-like”) properties in vitro, recurrently detected in genetically engineered mouse models of EAC and in EAC patient samples in vivo, and portended an overall poor prognosis. Collectively, these findings indicate that progression from chronic injury to malignancy in the esophagus is driven by a unique mechanism that links redox balance, inflammation, embryonic development (NOTCH) together into a common pro-tumorigenic pathway that is intrinsic to EAC cells. Citation Format: Lei Chen, Heng Lu, Dunfa Peng, Longlong Cao, Zheng Chen, Ajaz Bhat, Alexander Zaika, Shutian Zhang, Wael El-Rifai. Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6071.

Components of NOTCH Signaling for Uterine Cancer Patients' Prognosis.

New molecular biomarkers that could have an independent prognostic value in endometrial cancer are currently under investigation. Recently, it was suggested that genetic changes in the Notch signaling pathway could be associated with the development of endometrial carcinoma. This study aimed to determine the expression of the Notch signaling pathway components in tumour and adjacent normal uterine tissue and to evaluate their importance for the survival of uterine cancer patients. The present study was performed on uterine body samples collected from 109 patients and paired adjacent noncancerous endometrial tissue samples. Kaplan–Meier curves and Cox regression were used for survival analyses. Expression alterations of NOTCH2, NOTCH3, NOTCH4, JAG2, and HES1 were evaluated as independent and significant prognostic factors for uterine cancer patients.

Assessing the Roles of Potential Notch Signaling Components in Instructive and Permissive Pathways with Two Drosophila Pericardial Reporters.

The highly conserved Notch signaling pathway brings about the transcriptional activation of target genes via either instructive or permissive mechanisms that depend on the identity of the specific target gene. As additional components of the Notch signaling pathway are identified, assessing whether each of these components are utilized exclusively by one of these mechanisms (and if so, which), or by both, becomes increasingly important. Using RNA interference-mediated knockdowns of the Notch component to be tested, reporters for two Notch-activated pericardial genes in Drosophila melanogaster, immunohistochemistry, and fluorescence microscopy, we describe a method to determine the type of signaling mechanism-instructive, permissive, or both-to which a particular Notch pathway component contributes.

Notch signaling via Hey1 and Id2b regulates Müller glia's regenerative response to retinal injury.

Zebrafish Müller glia (MG) respond to retinal injury by suppressing Notch signaling and producing progenitors for retinal repair. A certain threshold of injury-derived signal must be exceeded in order to engage MG in a regenerative response (MG's injury-response threshold). Pan-retinal Notch inhibition expands the zone of injury-responsive MG at the site of focal injury, suggesting that Notch signaling regulates MG's injury-response threshold. We found that Notch signaling enhanced chromatin accessibility and gene expression at a subset of regeneration-associated genes in the uninjured retina. Two Notch effector genes, hey1 and id2b, were identified that reflect bifurcation of the Notch signaling pathway, and differentially regulate MG's injury-response threshold and proliferation of MG-derived progenitors. Furthermore, Notch signaling component gene repression in the injured retina suggests a role for Dll4, Dlb, and Notch3 in regulating Notch signaling in MG and epistasis experiments confirm that the Dll4/Dlb-Notch3-Hey1/Id2b signaling pathway regulates MG's injury-response threshold and proliferation.

Single-Cell RNA Sequencing Analysis of Chicken Anterior Pituitary: A Bird's-Eye View on Vertebrate Pituitary.

It is well-established that anterior pituitary contains multiple endocrine cell populations, and each of them can secrete one/two hormone(s) to regulate vital physiological processes of vertebrates. However, the gene expression profiles of each pituitary cell population remains poorly characterized in most vertebrate groups. Here we analyzed the transcriptome of each cell population in adult chicken anterior pituitaries using single-cell RNA sequencing technology. The results showed that: (1) four out of five known endocrine cell clusters have been identified and designated as the lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs, respectively. Somatotrophs were not analyzed in the current study. Each cell cluster can express at least one known endocrine hormone, and novel marker genes (e.g., CD24 and HSPB1 in lactotrophs, NPBWR2 and NDRG1 in corticotrophs; DIO2 and SOUL in thyrotrophs, C5H11ORF96 and HPGDS in gonadotrophs) are identified. Interestingly, gonadotrophs were shown to abundantly express five peptide hormones: FSH, LH, GRP, CART and RLN3; (2) four non-endocrine/secretory cell types, including endothelial cells (expressing IGFBP7 and CFD) and folliculo-stellate cells (FS-cells, expressing S100A6 and S100A10), were identified in chicken anterior pituitaries. Among them, FS-cells can express many growth factors, peptides (e.g., WNT5A, HBEGF, Activins, VEGFC, NPY, and BMP4), and progenitor/stem cell-associated genes (e.g., Notch signaling components, CDH1), implying that the FS-cell cluster may act as a paracrine/autocrine signaling center and enrich pituitary progenitor/stem cells; (3) sexually dimorphic expression of many genes were identified in most cell clusters, including gonadotrophs and lactotrophs. Taken together, our data provides a bird’s-eye view on the diverse aspects of anterior pituitaries, including cell composition, heterogeneity, cell-to-cell communication, and gene expression profiles, which facilitates our comprehensive understanding of vertebrate pituitary biology.