Repression of Sox9 by Jag1 is continuously required to suppress the default chondrogenic fate of vascular smooth muscle cells.

Acquisition and maintenance of vascular smooth muscle fate are essential for the morphogenesis and function of the circulatory system. Loss of contractile properties or changes in the identity of vascular smooth muscle cells (vSMCs) can result in structural alterations associated with aneurysms and vascular wall calcification. Here we report that maturation of sclerotome-derived vSMCs depends on a transcriptional switch between mouse embryonic days 13 and 14.

Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin.

Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis remains unresolved.

Optical tweezers studies on Notch: single-molecule interaction strength is independent of ligand endocytosis.

Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells but require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established.

Notch ligand endocytosis: mechanistic basis of signaling activity.

Regulation of Notch signaling is critical to development and maintenance of most eukaryotic organisms. The Notch receptors and ligands are integral membrane proteins and direct cell-cell interactions are needed to activate signaling. Ligand-expressing cells activate Notch signaling through an unusual mechanism involving Notch proteolysis to release the intracellular domain from the membrane, allowing the Notch receptor to function directly as the downstream signal transducer.

Notch ligand ubiquitylation: what is it good for?

In the first volume of Developmental Cell, it was reported that the classic Drosophila neurogenic gene neuralized encodes a ubiquitin ligase that monoubiquitylates the Notch ligand Delta, thus promoting Delta endocytosis. A requirement for ligand internalization by the signal-sending cell, although counterintuitive, remains to date a feature unique to Notch signaling. Ten years and many ubiquitin ligases later, we discuss sequels to these three papers with an eye toward reviewing the development of ideas for how ligand ubiquitylation and endocytosis propel Notch signaling.

Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome.

Mutations in the human Notch ligand jagged 1 (JAG1) result in a multi-system disorder called Alagille syndrome (AGS). AGS is chiefly characterized by a paucity of intrahepatic bile ducts (IHBD), but also includes cardiac, ocular, skeletal, craniofacial and renal defects. The disease penetration and severity of the affected organs can vary significantly and the molecular basis for this broad spectrum of pathology is unclear.

Canonical and non-canonical Notch ligands.

Notch signaling induced by canonical Notch ligands is critical for normal embryonic development and tissue homeostasis through the regulation of a variety of cell fate decisions and cellular processes. Activation of Notch signaling is normally tightly controlled by direct interactions with ligand-expressing cells, and dysregulated Notch signaling is associated with developmental abnormalities and cancer. While canonical Notch ligands are responsible for the majority of Notch signaling, a diverse group of structurally unrelated noncanonical ligands has also been identified that activate Notch and likely contribute to the pleiotropic effects of Notch signaling.

Osteogenic oxysterol, 20(S)-hydroxycholesterol, induces notch target gene expression in bone marrow stromal cells.

We previously reported that specific oxysterols stimulate osteogenic differentiation of pluripotent bone marrow stromal cells (MSCs) through activation of hedgehog (Hh) signaling and may serve as potential future therapies for intervention in osteopenia and osteoporosis. In this study we report that the osteogenic oxysterol 20(S)-hydroxycholesterol (20S) induces the expression of genes associated with Notch signaling. Using M2-10B4 (M2) MSCs, we found that 20S significantly induced HES-1, HEY-1, and HEY-2 mRNA expression compared with untreated cells, with maximal induction after 48 hours, whereas the nonosteogenic oxysterols did not.

Selective use of ADAM10 and ADAM17 in activation of Notch1 signaling.

Notch signaling requires a series of proteolytic cleavage events to release the Notch intracellular domain (NICD) that functions directly in signal transduction. The Notch receptor is locked down in a protease-resistant state by a negative regulatory region (NRR) that protects an ADAM (a disintegrin and metalloprotease) cleavage site. Engagement with ligand-bearing cells induces global conformational movements in Notch that unfold the NRR structure to expose the ADAM cleavage site and initiate proteolytic activation.

Numb regulates post-endocytic trafficking and degradation of Notch1.

Notch is a transmembrane receptor that controls cell fate decisions during development and tissue homeostasis. Both activation and attenuation of the Notch signal are tightly regulated by endocytosis. The adaptor protein Numb acts as an inhibitor of Notch and is known to function within the intracellular trafficking pathways.

The many facets of Notch ligands.

The Notch signaling pathway regulates a diverse array of cell types and cellular processes and is tightly regulated by ligand binding. Both canonical and noncanonical Notch ligands have been identified that may account for some of the pleiotropic nature associated with Notch signaling. This review focuses on the molecular mechanisms by which Notch ligands function as signaling agonists and antagonists, and discusses different modes of activating ligands as well as findings that support intrinsic ligand signaling activity independent of Notch.

Notch signaling--constantly on the move.

The Notch pathway is a highly conserved and ubiquitous signaling system that functions in determining a diverse array of cell fates and regulates many cellular processes during embryonic development and throughout adulthood. Links to cancer, stroke and Alzheimer's disease underscore the need to define the molecular basis of Notch activation. Notch signaling is induced through direct cell-cell interactions that promote receptor activation following engagement with a membrane-bound Delta, Serrate, Lag-2 (DSL) ligand on adjacent cells.

DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur.

Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity.

A garden of Notch-ly delights.

Over the past decade, the Notch signaling pathway has been shown to be crucially important for normal metazoan development and to be associated with several human inherited and late onset diseases. The realization that altered Notch signaling contributes at various levels to human disease lead in May to the first meeting dedicated solely to Notch signaling in vertebrate development and disease in Madrid, Spain. Hosted by the Cantoblanco Workshops on Biology and organized by Tom Gridley, José Luis de la Pompa and Juan Carlos Izpisúa Belmonte, the meeting covered diverse aspects of this important signaling pathway.

Microfibrillar proteins MAGP-1 and MAGP-2 induce Notch1 extracellular domain dissociation and receptor activation.

Unlike most receptors, Notch serves as both the receiver and direct transducer of signaling events. Activation can be mediated by one of five membrane-bound ligands of either the Delta-like (-1, -2, -4) or Jagged/Serrate (-1, -2) families. Alternatively, dissociation of the Notch heterodimer with consequent activation can also be mediated experimentally by calcium chelators or by mutations that destabilize the Notch1 heterodimer, such as in the human disease T cell acute lymphoblastic leukemia.

The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands.

Mutations in the DSL (Delta, Serrate, Lag2) Notch (N) ligand Delta-like (Dll) 3 cause skeletal abnormalities in spondylocostal dysostosis, which is consistent with a critical role for N signaling during somitogenesis. Understanding how Dll3 functions is complicated by reports that DSL ligands both activate and inhibit N signaling. In contrast to other DSL ligands, we show that Dll3 does not activate N signaling in multiple assays.

Functional analysis of a recurrent missense mutation in Notch3 in CADASIL.

Background: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular dementia characterised by recurrent ischemic strokes in the deep white matter. Mutations in the gene encoding the cell surface receptor, Notch3, have been identified in CADASIL patients, and accumulation of the extracellular domain of Notch3 has been demonstrated in affected vessels. Almost all CADASIL mutations alter the number of cysteine residues in the epidermal growth factor (EGF)-like repeats in the extracellular domain of the protein.

The extracellular matrix protein MAGP-2 interacts with Jagged1 and induces its shedding from the cell surface.

Elastic fibers are composed of the protein elastin and a network of 10-12-nm microfibrils, which are composed of several glycoproteins, including fibrillin-1, fibrillin-2, and MAGP1/2 (microfibril-associated glycoproteins-1 and -2). Although fibrillins and MAGPs covalently associate, we find that the DSL (Delta/Serrate/LAG2) protein Jagged1, an activating ligand for Notch receptor signaling, also interacts with MAGP-2 in both yeast two-hybrid and coimmunoprecipitation studies. Interaction between Jagged1 and MAGP-2 requires the epidermal growth factor-like repeats of Jagged1.

Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1.

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells.

Modulation of notch signaling during somitogenesis.

The Notch signaling pathway is known to govern various aspects of tissue differentiation during embryonic development by mediating local cell-cell interactions that often control cell fate. The conserved components that underlie Notch signaling have been isolated in vertebrates, leading to a biochemical delineation of a core Notch signaling pathway and functional studies of this pathway during embryogenesis. Herein we highlight recent progress in determining how Notch signaling contributes to the development of the vertebrate embryo.

Extrinsic and intrinsic factors governing cell fate in cortical progenitor cultures.

Central nervous system germinal zones contain stem cells that generate both neurons and glia. In the recent past, these cells have been isolated, maintained in a variety of culture systems and used in vitro for subsequent characterization of molecular mechanisms underlying brain development. Factors that govern cell fate choices of these neural stem cells have not been fully elucidated, but recent studies suggest that age at the time of culture is an important intrinsic mechanism.

Sequential signaling through Notch1 and erbB receptors mediates radial glia differentiation.

Radial glia cells both generate neurons and physically guide nascent neurons to their target destination in the cortex, and as such they are essential for CNS development. It has been proposed that in the developing cerebellum, neuronal contact induces radial glia formation, however, the mechanisms involved in this process are not well understood. Here we demonstrate that neuronal induction of radial glia formation is the result of sequential signaling through Notch1 and erbB receptors.

Basal expression of IkappaBalpha is controlled by the mammalian transcriptional repressor RBP-J (CBF1) and its activator Notch1.

By using the hepatic stellate cell (HSC) as a paradigm for cells that undergo long term re-programming of NF-kappaB-dependent transcription, we have determined a novel mechanism by which mammalian cells establish their basal NF-kappaB activity. Elevation of NF-kappaB activity during HSC activation is accompanied by induction of CBF1 expression and DNA binding activity. We show that the transcriptional repressor CBF1 interacts with a dual NF-kappaB/CBF1-binding site (kappaB2) in the IkappaBalpha promoter.

Glycoprotein 130 signaling regulates Notch1 expression and activation in the self-renewal of mammalian forebrain neural stem cells.

Glycoprotein130 (gp130) and Notch signaling are thought to participate in neural stem cell (NSC) self-renewal. We asked whether gp130 regulates Notch activity in forebrain epidermal growth factor (EGF)-responsive NSCs. Disruption of Notch1 using antisense or a gamma-secretase inhibitor demonstrated a requirement for Notch1 in the maintenance and proliferation of NSCs.

Modulation of the notch signaling by Mash1 and Dlx1/2 regulates sequential specification and differentiation of progenitor cell types in the subcortical telencephalon.

Notch signaling has a central role in cell fate specification and differentiation. We provide evidence that the Mash1 (bHLH) and Dlx1 and Dlx2 (homeobox) transcription factors have complementary roles in regulating Notch signaling, which in turn mediates the temporal control of subcortical telencephalic neurogenesis in mice. We defined progressively more mature subcortical progenitors (P1, P2 and P3) through their combinatorial expression of MASH1 and DLX2, as well as the expression of proliferative and postmitotic cell markers at E10.