Notch-activated Cells Research Articles

Changes in searching behaviour of CSL transcription complexes in Notch active conditions.

During development cells receive a variety of signals, which are of crucial importance to their fate determination. One such source of signal is the Notch signalling pathway, where Notch activity regulates expression of target genes through the core transcription factor CSL. To understand changes in transcription factor behaviour that lead to transcriptional changes in Notch active cells, we have probed CSL behaviours in real time, using in vivo Single Molecule Localisation Microscopy. Trajectory analysis reveals that Notch-On conditions increase the fraction of bound CSL molecules, but also the proportion of molecules with exploratory behaviours. These properties are shared by the co-activator Mastermind. Furthermore, both CSL and Mastermind, exhibit characteristics of local exploration near a Notch target locus. A similar behaviour is observed for CSL molecules diffusing in the vicinity of other bound CSL clusters. We suggest therefore that CSL acquires an exploratory behaviour when part of the activation complex, favouring local searching and retention close to its target enhancers. This change explains how CSL can efficiently increase its occupancy at target sites in Notch-On conditions.

Jagged 2b induces intercellular signaling within somites to establish hematopoietic stem cell fate in zebrafish.

During development, the somites play a key role in the specification of hematopoietic stem cells (HSCs). In zebrafish, the somitic Notch ligands Delta-c (Dlc) and Dld, both of which are regulated by Wnt16, directly instruct HSC fate in a shared vascular precursor. However, it remains unclear how this signaling cascade is spatially and temporally regulated within somites. Here, we show in zebrafish that an additional somitic Notch ligand, Jagged 2b (Jag2b), induces intercellular signaling to drive wnt16 expression. Jag2b activated Notch signaling in segmented somites at the early stage of somitogenesis. Loss of jag2b led to a reduction in the expression of wnt16 in the somites and an HSC marker, runx1, in the dorsal aorta, whereas overexpression of jag2b increased both. However, Notch-activated cells were adjacent to, but did not overlap with, wnt16-expressing cells within the somites, suggesting that an additional signaling molecule mediates this intercellular signal transduction. We uncover that Jag2b-driven Notch signaling induces efna1b expression, which regulates wnt16 expression in neighboring somitic cells. Collectively, we provide evidence for previously unidentified spatiotemporal regulatory mechanisms of HSC specification by somites.

Retinal Wnt signaling defect in a zebrafish fetal alcohol spectrum disorder model.

Fetal alcohol spectrum disorder caused by prenatal alcohol exposure includes ocular abnormalities (microphthalmia, photoreceptor dysfunction, cataracts). Zebrafish embryos exposed to ethanol from gastrulation through somitogenesis show severe ocular defects, including microphthalmia and photoreceptor differentiation defects. Ethanol-treated zebrafish had an enlarged ciliary marginal zone (CMZ) relative to the retina size and reduced Müller glial cells (MGCs). Ethanol exposure produced immature photoreceptors with increased proliferation, indicating cell cycle exit failure. Signaling mechanisms in the CMZ were affected by embryonic ethanol exposure, including Wnt signaling in the CMZ, Notch signaling and neurod gene expression. Retinoic acid or folic acid co-supplementation with ethanol rescued Wnt signaling and retinal differentiation. Activating Wnt signaling using GSK3 inhibitor (LSN 2105786; Eli Lilly and Co.) restored retinal cell differentiation pathways. Ethanol exposed embryos were treated with Wnt agonist, which rescued Wnt-active cells in the CMZ, Notch-active cells in the retina, proliferation, and photoreceptor terminal differentiation. Our results illustrate the critical role of Wnt signaling in ethanol-induced retinal defects.

Use of Hes1-GFP reporter mice to assess activity of the Hes1 promoter in bone cells under chronic inflammation

Notch signaling plays a critical role in maintaining bone homeostasis partially by controlling the formation of osteoblasts from mesenchymal stem cells (MSCs). We reported that TNF activates Notch signaling in MSCs which inhibits osteoblast differentiation in TNF transgenic (TNF-Tg) mice, a mouse model of chronic inflammatory arthritis. In the current study, we used Hes1-GFP and Hes1-GFP/TNF-Tg mice to study the distribution and dynamic change of Notch active cells in normal and inflammatory bone loss and mechanisms mediating their enhanced proliferation. We found that Hes1-GFP+ cells are composed of cells expressing mesenchymal, hematopoietic and endothelial surface markers. CD45−/Hes1-GFP+ cells express high levels of mesenchymal markers and form CFU-F and CFU-ALP colonies. Expansion of CFU-F colonies is associated with a rapid increase in Hes1-GFP+ cell numbers and their GFP intensity. The GFP signal is lost when a CFU-F colony differentiates into an ALP+ osteoblast colony. TNF increases the numbers of CD45−/Hes1-GFP+ cells, which are stained negatively for osteoblast marker osteocalcin and localized adjacent to endosteal and trabecular bone surfaces. CD45−/Hes1-GFP+ cells in Hes1-GFP/TNF-Tg mice have increased BrdU incorporation and PDGFRβ levels. TNF increases the number of proliferating Hes1-GFP+ cells, which is prevented by a specific PDGFRβ inhibitor. Notch inhibition blocks TNF-mediated PDGFRβ expression and cell proliferation. Thus, TNF-induced MSC proliferation is mediated by PDGFRβ signal, which works at downstream of Notch. Hes1-GFP mice can be used to assess the activation status of Notch in bone cells.

Abstract A37: Tumor heterogeneity in SCLC: A role for endogenous Notch signaling

Abstract Small cell lung cancer (SCLC), a very aggressive neuroendocrine (NE) tumor of the lung, constitutes approximately 15% of all lung cancers and has a 5-year survival rate of only 5%. Resistance following initial chemotherapy and radiation treatments accounts for the high rate of recurrence and the overall poor prognosis of SCLC. Through genomic sequencing of 100 human SCLC tumors, the group of Roman Thomas and Martin Peifer identified universal inactivation of RB and p53 and inactivating mutations in the NOTCH receptors in ~25% of the tumors. Accordingly, we found that activation of the Notch pathway in a pre-clinical mouse model for SCLC dramatically reduces the number of tumors and extends the survival of the mice. Acute Notch activation also inhibits NE gene expression and proliferation in SCLC tumor cells, providing further evidence for a tumor suppressive role for Notch in SCLC (George, Lim, et al., Nature, 2015). Intriguingly, however, we recently identified a population of cells in mouse and human SCLC tumors that exhibit endogenous activation of the Notch pathway. This observation raises the question of why tumor cells would activate a tumor suppressive pathway. Consistent with the inhibitory role of the pathway in NE differentiation, these Notch-active SCLC cells comprise a slow-cycling, non-NE compartment within SCLC tumors. Lineage tracing experiments indicate that NE tumor cells generate these non-NE Notch-active cells during tumorigenesis. Importantly, our data suggest that these slow-growing Notch-active SCLC cells may function as tumor-derived stromal-like cells that confer a growth or protective advantage to the NE tumor cells under certain growth conditions. In conclusion, Notch is a potent suppressor of SCLC. However, Notch may have pro-tumorigenic effects in SCLC, possibly under stress conditions. A better understanding of the molecular and cellular roles of Notch is required before therapeutic strategies manipulating the Notch pathway can be used in SCLC patients. Citation Format: Jing Shan Lim, Alvaro Ibaseta, Dian Yang, Nadine S. Jahchan, Julien Sage. Tumor heterogeneity in SCLC: A role for endogenous Notch signaling. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A37.

An integrated genomic approach identifies that the PI3K/AKT/FOXO pathway is involved in breast cancer tumor initiation

Therapy resistance is one of the major impediments to successful cancer treatment. In breast cancer, a small subpopulation of cells with stem cell features, named breast cancer stem cells (BCSC), is responsible for metastasis and recurrence of the tumor. BCSC have the unique ability to grow under non-adherent conditions in “mammospheres”. To prevent breast cancer recurrence and metastasis it will be crucial to eradicate BCSC.We used shRNA genetic screening to identify genes that upon knockdown enhance mammosphere formation in breast cancer cells. By integration of these results with gene expression profiles of mammospheres and NOTCH-activated cells, we identified FOXO3A. Modulation of FOXO3A activity results in a change in mammosphere formation, expression of mammary stem cell markers and breast cancer initiating potential. Importantly, lack of FOXO3A expression in breast cancer patients is associated with increased recurrence rate. Our findings provide evidence for a role for FOXO3A downstream of NOTCH and AKT that may have implications for therapies targeting BCSCs.

Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas

The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

Notch activation promotes endothelial survival through a PI3K-Slug axis

RationaleLoss of endothelial viability correlates with initiation and progress of vascular pathology. However, much remains to be learned about pathways required to maintain the balance between cell viability and apoptosis. Notch activation can enhance or inhibit apoptosis but its role in maintaining the endothelium needs further delineation. ObjectiveThis study aims to identify the mechanisms by which Notch activation regulates endothelial viability. Methods and resultsEndothelial cells transduced with active Notch were treated with lipopolysaccharide (LPS) or homocysteine to induce endothelial apoptosis. Notch protected against LPS-induced cell death but exacerbated homocysteine-induced apoptosis. Inhibition of PI3K revealed that ligand-induced activation of endogenous Notch initiates parallel death and survival pathways and exhibits a differential effect on endothelial survival depending on the apoptotic stimulus. PI3K activity regulated the expression of Slug, which was required for survival in Notch-activated endothelial cells. Homocysteine, but not LPS, blocked both PI3K activity and Slug expression in Notch-activated cells, leading to increased endothelial apoptosis. ConclusionsNotch signaling leads to activation of parallel survival and apoptotic pathways in endothelial cells. The interaction of Notch with other signaling pathways plays an important contextual role in regulating endothelial viability.

Reprogramming the Beat

The explosion of stem cell research in cardiology has yielded an increasing recognition that understanding developmental cell fate decisions is critical for everything from disease models to cellular therapeutics. However, the biology is particularly rich and complex and has not yielded easily to traditional investigative methods. One area of intense focus is the study of the cues responsible for the development of specialized conduction tissues in the heart. Direction of native cells or exogenous cells into the conduction system lineage might offer therapeutic insights into degenerative conduction disease. Article see p 1058 The most distal subendocardial fibers of the cardiac conduction system were first described by Jan Purkinje in 1839.1 Over 100 years ago, in painstakingly delicate dissections, Sunao Tawara2 characterized the cardiac conduction system further, describing the atrioventricular node, the penetrating bundle of His, the bundle branches, and the Purkinje fiber network. During the past 20 years, however, the pace of discovery of this highly specialized tissue has accelerated. Beginning with the observations that peripheral and central conduction tissue derives from cardiomyocyte progenitors in the chick,3,4 investigators have now identified key roles for many important molecular signals and transcription factors in conduction system development. Signaling molecules implicated in the formation of the conduction system include endothelin,5–9 neuregulin,10–13 Wnts,14 and bone morphogenetic proteins,15 and complex transcriptional roles have been identified for NKx2.5,16–20 members the T-box transcription factor family,18,21,22 shox2,23 msx2,24,25 and hop.26–28 In this issue of Circulation , Rentschler et al29 take a significant step toward understanding the determinants of conduction cell fate. In a series of elegant experiments, they demonstrate that activation of …

Notch Initiates the Endothelial-to-Mesenchymal Transition in the Atrioventricular Canal through Autocrine Activation of Soluble Guanylyl Cyclase

The heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC).

Abstract 3352: Therapeutic targeting of the Notch pathway in breast cancer stem cells

Abstract Recent identification of breast cancer stem cells (CSCs) suggest that a subpopulation of tumor cells with a stem cell phenotype may initiate and maintain tumors. Breast CSCs are characterized by their ability to self-renew and generate tumors in serial xenotransplantation assays in mice. The Notch pathway is an evolutionally conserved signaling pathway and Notch activity has been implicated in normal and malignant mammary stem cell maintenance. Therefore, we investigated the role of Notch in breast CSCs by utilizing a number of CSC specific assays and a mouse xenograft model. Upon activation, the Notch intracellular domain (NICD) is cleaved by the gamma secretase complex and translocates to the nucleus. Subsequently, it binds to the transcriptional complex (RBP-jk), to activate target genes such as the HES and HEY family of proteins. The purpose of this study is to test the Notch activity in CSCs from breast cancer cell lines including basal, luminal, and HER2-amplified subtypes. We hypothesize that cells with higher Notch activity may be enriched for cells with stem cell properties. To study the Notch pathway in breast cancer cell lines we used a Notch reporter that contains multiple copies of the Notch response elements upstream of the mCMV promoter that turns on GFP and luciferase expression upon Notch activation. We measured Notch activity by flow cytometry of cells or by bioluminescent imaging of tumors in mice utilizing Notch responsive GFP and luciferase expression, respectively. In addition, we utilized a gamma secretase inhibitor (GSI) which inhibits the cleavage of NICD and nuclear translocation. We treated mice with GSI, docetaxel, or GSI and docetaxel in combination. We have found that basal cell lines such as Sum 159 and MDA-MB-436 have a higher expression of Notch activity compared to the ER+ luminal cell lines MCF7, T47D, and ZR-75-1. We demonstrated that Notch activated cells show a stem cell phenotype with co-localization of expression of the Notch target gene HES1 and the stem cell marker ALDH1. In addition, Notch active cells were able to generate tumors in serial xenotransplantations in mice and to generate both Notch GFP-positive and Notch-GFP negative cells in vitro and in vivo. In contrast, Notch-GFP negative cells neither gave rise to Notch-GFP positive cells nor did they generate tumors in serial xenotransplantation assays. GSI treatment of mice with breast tumors decreased the percentage of Notch-GFP positive cells in the tumor, reducing the cancer stem cell population and decreasing tumorigenesis in serial passages. Furthermore, the combination of docetaxel with GSI further reduced tumor size by targeting the bulk tumor cell populations. These studies strongly suggest that the Notch pathway plays an essential role in breast CSC maintenance and may be an attractive target for more effective treatment. The combination therapy of GSI with cytotoxic chemotherapy is currently being evaluated in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3352. doi:10.1158/1538-7445.AM2011-3352

Notch-GATA synergy promotes endoderm-specific expression ofref-1inC. elegans

The Notch signaling pathway is involved in a wide variety of cell-fate decisions during development. The diverse behavior of Notch-activated cells is thought to depend on tissue- or cell-type-specific transcription factors, yet the identities of such factors and the mechanism of cooperation with the Notch pathway are largely unknown. We identify here an enhancer in the promoter of ref-1, a C. elegans Notch target, which promotes Notch-dependent expression in mesodermal and endodermal cells. The enhancer contains predicted binding sites for the Notch transcriptional effector LAG-1/CSL that are essential for expression, a non-CSL site required for mesodermal expression, and four predicted binding sites for GATA transcription factors that are required for endodermal expression. We show that endodermal expression involves the GATA transcription factor ELT-2, and that ELT-2 can bind LAG-1/CSL in vitro. In many types of Notch-activated embryonic cells, ectopic ELT-2 is sufficient to drive expression of reporters containing the enhancer.

Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development

During development of the chicken proventriculus (glandular stomach), gut endoderm differentiates into glandular and luminal epithelium. We found that Delta1-expressing cells, undifferentiated cells and Notch-activated cells colocalize within the endodermal epithelium during early gland formation. Inhibition of Notch signaling using Numb or dominant-negative form of Su(H) resulted in a luminal differentiation, while forced activation of Notch signaling promoted the specification of immature glandular cells, but prevented the subsequent differentiation and the invagination of the glands. These results suggest that Delta1-mediated Notch signaling among endodermal cells functions as a binary switch for determination of glandular and luminal fates, and regulates patterned differentiation of glands in the chicken proventriculus.