Notch activity acts as a sensor for extracellular calcium during vertebrate left-right determination

During vertebrate embryo development, the breaking of the initial bilateral symmetry is translated into asymmetric gene expression around the node and/or in the lateral plate mesoderm. The earliest conserved feature of this asymmetric gene expression cascade is the left-sided expression of Nodal, which depends on the activity of the Notch signalling pathway. Here we present a mathematical model describing the dynamics of the Notch signalling pathway during chick embryo gastrulation, which reveals a complex and highly robust genetic network that locally activates Notch on the left side of Hensen's node. We identify the source of the asymmetric activation of Notch as a transient accumulation of extracellular calcium, which in turn depends on left-right differences in H+/K+-ATPase activity. Our results uncover a mechanism by which the Notch signalling pathway translates asymmetry in epigenetic factors into asymmetric gene expression around the node.     

Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase

Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.     

Notch signalling controls pancreatic cell differentiation.

The pancreas contains both exocrine and endocrine cells, but the molecular mechanisms controlling the differentiation of these cell types are largely unknown. Despite their endodermal origin, pancreatic endocrine cells share several molecular characteristics with neurons, and, like neurons in the central nervous system, differentiating endocrine cells in the pancreas appear in a scattered fashion within a field of progenitor cells. This indicates that they may be generated by lateral specification through Notch signalling. Here, to test this idea, we analysed pancreas development in mice genetically altered at several steps in the Notch signalling pathway. Mice deficient for Delta-like gene 1 (Dll1) or the intracellular mediator RBP-Jkappa showed accelerated differentiation of pancreatic endocrine cells. A similar phenotype was observed in mice over-expressing neurogenin 3 (ngn 3) or the intracellular form of Notch3 (a repressor of Notch signalling). These data provide evidence that ngn3 acts as proendocrine gene and that Notch signalling is critical for the decision between the endocrine and progenitor/exocrine fates in the developing pancreas.     

Sustained translational repression by eIF2α-P mediates prion neurodegeneration

The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer's, Parkinson's and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer's, Parkinson's and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation, increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.     

Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants

Presenilin proteins have been implicated both in developmental signalling by the cell-surface protein Notch and in the pathogenesis of Alzheimer's disease. Loss of presenilin function leads to Notch/lin-12-like mutant phenotypes in Caenorhabditis elegans and to reduced Notch1 expression in the mouse paraxial mesoderm. In humans, presenilins that are associated with Alzheimer's disease stimulate overproduction of the neurotoxic 42-amino-acid beta-amyloid derivative (Abeta42) of the amyloid-precursor protein APP. Here we describe loss-of-function mutations in the Drosophila Presenilin gene that cause lethal Notch-like phenotypes such as maternal neurogenic effects during embryogenesis, loss of lateral inhibition within proneural cell clusters, and absence of wing margin formation. We show that presenilin is required for the normal proteolytic production of carboxy-terminal Notch fragments that are needed for receptor maturation and signalling, and that genetically it acts upstream of both the membrane-bound form and the activated nuclear form of Notch. Our findings provide evidence for the existence of distinct processing sites or modifications in the extracellular domain of Notch. They also link the role of presenilin in Notch signalling to its effect on amyloid production in Alzheimer's disease.     

Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.     

Fringe is a glycosyltransferase that modifies Notch

Notch receptors function in highly conserved intercellular signalling pathways that direct cell-fate decisions, proliferation and apoptosis in metazoans. Fringe proteins can positively and negatively modulate the ability of Notch ligands to activate the Notch receptor. Here we establish the biochemical mechanism of Fringe action. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. We obtained biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signalling by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signalling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signalling pathways.     

A somitic Wnt16/Notch pathway specifies haematopoietic stem cells

Haematopoietic stem cells (HSCs) are a self-renewing population of cells that continuously replenish all blood and immune cells during the lifetime of an individual. HSCs are used clinically to treat a wide array of diseases, including acute leukaemias and congenital blood disorders, but obtaining suitable numbers of cells and finding immune-compatible donors remain serious problems. These difficulties have led to an interest in the conversion of embryonic stem cells or induced pluripotent stem cells into HSCs, which is not possible using current methodologies. To accomplish this goal, it is critical to understand the native mechanisms involved in the specification of HSCs during embryonic development. Here we demonstrate in zebrafish that Wnt16 controls a novel genetic regulatory network required for HSC specification. Non-canonical signalling by Wnt16 is required for somitic expression of the Notch ligands deltaC (dlc) and deltaD (dld), and these ligands are, in turn, required for the establishment of definitive haematopoiesis. Notch signalling downstream of Dlc and Dld is earlier than, and distinct from, known cell-autonomous requirements for Notch, strongly suggesting that novel Notch-dependent relay signal(s) induce the first HSCs in parallel to other established pathways. Our results demonstrate that somite-specific gene expression is required for the production of haemogenic endothelium.     

A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain

Signalling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologues in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signalling pathway. Here we report that, in mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signalling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signalling.