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Signalling: 1 Wnt | 2 Notch | 3 FGF Receptor | 4 Hedgehog | 5 T-box | 6 TGF-Beta
2016 Group Project Topic - Signaling in Development

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The Notch Signalling Pathway

Introduction

The Notch signalling pathway is the most prevalent form of intracellular communication in multicellular organisms. It is a critical pathway for cell differentiation, proliferation, and apoptosis. It is involved in embryonic organ development through the regulation of cell-cell signalling; specifically lateral inhibition, formation of boundaries, and cell lineage assignation.[1][2] This pathway is also actively involved in adjacent cell communication, developmental process such as adult homeostasis, and stem cell maintenance. Since this pathway is an intricate and crucial aspect of an organism's development and cell signalling, a mutation in the functional components of this pathway can cause a myriad of diseases such as congenital disorders, cancers, strokes, Alagille syndrome, and leukoencephalopathy.[3] The process of Notch signalling primarily utilises a ligand-receptor interaction to release protein fragments at the cellular membrane, which then provide signals to the internal environment of the cell and to adjacent cells. In mammals, the Notch signalling pathway is comprised of four receptors (Notch 1-4) which interact with Delta or Jagged ligands to bring about the activation of this pathway. Homologous Notch genes have been identified in other animals, such as Drosophila melanogaster (fruit fly) and Danio rerio (zebrafish).

What will you find on this wiki page?

This page is designed to provide an overview of such features of the Notch signalling pathway, but is in no way a complete resource for all Notch-related information (especially since this signalling pathway has such a wide array of activities in many organisms and the scientific understanding of it is continuously updating!). By reading this page you should come to a better understanding of Notch in regards to: general molecular mechanisms; embryonic development by systems; examples of animal development; abnormalities and disease; current and potential future research; as well as a couple of links to interesting articles for those who want to read more. There is a Glossary at the bottom of the page for better understanding of scientific terms used throughout this page, and readers are encouraged to 'expand' any collapsed information on the page to optimise their experience in reading about Notch.

<html5media width="560" height="315">https://www.youtube.com/embed/axkDX0XZyN0</html5media>


History

1914 First description of a "notch" defect (loss of tissue in the wing) in Drosophila by John S. Dexter, giving the gene its name[4]
1917 First allele of Notch is identified by Thomas Hunt Morgan[5]
1930s Donald F. Poulson conducts research into the involvement of Notch in development.[6]
1958 An investigation by W.J. Welshons confirmed that the locus of Notch is contained within the 3C7 band of the X chromosome.[7]
1983 DNA sequences belonging to the Notch locus cloned and determined that RNA is required for the function of the wild type Notch. Further insight developed into the function of Notch and its role in differentiation and regeneration.[8]
1986 The molecular analysis and sequence of the Notch locus was determined by Michael W. Young and his team. A relationship was identified between the protein encoded by the major Notch transcript and mammalian clotting and growth factors.[9]
1989 Neurogenic loci "Delta" and "Mastermind" identified and research conducted to search for genes which may have an interaction with the Notch protein. It was confirmed that mutations in these loci will affect neurogenesis.[10]
2012 The Notch ligand Jag1 was expressed in PA6 cells and showed demonstrated that the ability for the cells to differentiate was interrupted by the Notch signalling inhibition pathway. [11]
2014 Breakthrough in the applications of the Notch signalling pathway. Evidence emerged showing an association between the Notch signalling pathway and gastric cancer and found that Notch1 and Notch2 pathways have been activated in gastric cancer.[12]

Overview of Molecular Mechanisms

Mammals possess a total of four Notch genes, with five genes for encoding the associated ligands, Delta-like and Jagged. The Notch genes each code for a single transmembrane receptor. Extracellularly, it contains epidermal growth factor (EGF)-like repeats for ligand interaction and Lin-12-Notch (LN) repeats for regulating the interactions between the extracellular and intracellular regions. Intracellularly, Notch has seven ankyrin (ANK) repeats and a transactivation domain (TAD), as well as a proline, glutamine, serine, threonine-rich (PEST) domain for degradation of Notch.

The structure of the Notch receptor.[3] Extracellularly there are epidermal growth factor (EGF)-like repeats and Lin-12-Notch repeats (LNRs). The Notch intracellular domain (NICD) is made up of a rRBP-Jkappa-associated module (RAM) domain, ankyrin (ANK) repeats, and a proline, glutamine, serine, threonine-rich (PEST) domain. There are three sites for cleavage by enzymes: S1, S2, and S3/S4.

Canonical pathway

Summary of canonical Notch signalling. The interaction between Delta-like or Serrate ligands (DSL) and the Notch receptor on an adjacent cell initiates proteolytic cleavage of Notch and the release of the Notch intracellular domain (NICD). The NICD is then transported to the nucleus where it can induce transcription of Notch target genes by binding to specific proteins (MAM, CSL).[13]

The canonical Notch pathway is unique in that it involves direct interaction between adjacent cells, as opposed to paracrine signalling, because both the Notch ligands and Notch receptors are transmembrane proteins found in the cell membrane. Furthermore, the lack of a secondary messenger or amplification process means that the Notch pathway has limited opportunities for regulation and must therefore be tightly controlled. Depending on its developmental and cellular context, activation or inhibition of the pathway can result in a variety of cellular responses, including cell death, proliferation, or differentiation.[14]

Four Notch proteins are involved in the canonical pathway. NOTCH1 to NOTCH4 are single transmembrane receptors and can interact with a variety of ligands, including NOTCH ligands (e.g. Delta ligands) and Serrate ligands. There are three Delta ligands (Dll1, Dll3, and Dll4) and two Serrate ligands (Jagged1 and Jagged2) present in mammals.[14] The binding between the Notch receptor and the ligand on the adjacent cell induces the release of the Notch intracellular domain (NICD) via a sequence of proteolytic reactions.[1] Cell-cell interaction is therefore critical in the process of triggering Notch signalling.

The interaction on the cell surface between Notch and its ligand on an adjacent cell causes the extracellular metalloprotease site (S2 site) to be exposed. The S2 site is then cleaved by transmembrane proteases belonging to the a disintegrin and metalloproteinase/tumour necrosis factor α converting enzyme (ADAM/TACE) family. The remaining Notch fragment subsequently undergoes two more intramembranous cleavages at the S3/S4 sites by the γ-secretase complex. Finally, the NICD is released and enters the nucleus to interact with CSL (CBF1, Suppressor of Hairless, Lag-1) and Mastermind-like proteins (MAMLs). CSL is a DNA-binding protein that acts as a transcription factor by forming repressor or activator complexes; in the absence of the NCID, CSL is bound to corepressor proteins (CoR) that prevent transcription of Notch target genes. MAMLs are transcriptional co-activators that are required for transcription of the target genes, hence they are involved in the regulation of the pathway.[2][14] To stop signalling, the NICD is phosphorylated by kinases and ubiquitinated by E3 ubiquitin ligases, which results in proteasome mediated degradation and subsequent termination of the signal.[14]

Non-canonical pathway

Immense research has been carried out on the canonical Notch pathway and its members are well known. While canonical notch ligands control most of the known Notch signalling, a group of structurally distinct non-canonical ligands exist which activate Notch and its pleiotropic effects. Notch can non-canonically carry out its functions by post-translationally targeting Wnt/β-catenin signalling. This type of signalling is CSL-independent and can also work independently of ligands. Some genes are affected by the non-canonical Notch signalling, however their mediators in most cases are unknown. Notch binds and titrates levels of active β-catenin. Therefore, active β-catenin activity may serve as a useful readout for non-canonical Notch signals.[15]

Recent research has shown that Notch signalling plays crucial roles for cellular differentiation during development through γ-secretase-dependent intramembrane proteolysis followed by transcription of target genes. Hayashi et al. (2016) uncovered a ligand-dependent but γ-secretase-independent, non-canonical Notch signalling involved in presynaptic protein expression in postmitotic neurons.[16]

Examples of Non-Canonical Notch Signalling (adapted from Table 1[15])
Species Cell type Independent of Function Interacting molecule/signalling pathway
Human Human embryonic stem cells (in vitro) Ligand, CSL Negative regulation of Wnt signalling Active β-catenin/Wnt signalling
Rodent Mouse embryonic stem cells, neural stem cells, mesenchymal stem cells, cardiac progenitor cells Ligand, CSL Negative regulation of Wnt signalling Active β-catenin/Wnt signalling
T-cells CSL Notch1 stimulates NF-ϰb NF-ϰb pathway
Primary embryonic cells Presenilin, Ligand HES1 activation and MCK inhibition HES1 and MCK
Skin progenitor cells CSL Leukocytosis, longetivity
Avian Neural crest (stem cells) CSL Slug expression Slug
Frog Embryo CSL Negative regulation of Wnt signalling β-catenin/Wnt signalling
Fly Wing primordium Ligand, CSL Negative regulation of Wnt signalling Active β-catenin/Wnt signalling
CSL Inhibition of ligand function Serrate
Muscle progenitor cells Ligand, CSL Muscle precursor selection Wnt signalling
Embryo CSL Dorsal epidermis patterning (closure) JNK pathway
Neural precursors CSL Repression of neural fate Wnt signalling


Transcriptional regulation of Notch signalling

A number of genes have been found to act as modulators of Notch signalling by modifying the ability of Notch to interact with its ligands. These include proteins acting extracellularly (Fringe, Brainiac, Egghead, Scabrous, Wingless), proteins acting at the cell membrane (Big brain), proteins acting intracellularly (Numb, Sanpodo, Disabled, Deltex, Dishevelled), and proteins acting within the nucleus (Hairless, EMB-5, Strawberry notch).[17]

In Drosophila melanogaster, it has been seen that fringe is able to affect the ability of Notch to be activated by the Serrate and Delta ligands. Specifically in the Drosophila wing, it was observed that Serrate did not interact with Notch in specific areas due to the action of fringe[18]. Fringe is not required for initial Notch signalling, but is thought to be important for signalling processes when Notch activation occurs along the borders between distinct cell populations. Research suggests that Fringe specifically affects the binding between Notch and its ligands, rather than an activation step separate or subsequent to ligand binding. The brainiac and egghead genes are thought to either influence the production of a Notch ligand or act as substitute ligands themselves.[17]

Examples of Proteins Involved in Interaction with the Notch Intracellular Domain (adapted from Table 3[19])

Protein Interaction with NICD
Adenamatous polyposis coli (Apc) Controls Notch trafficking
Cyclin-dependant kinase 8 (CDK8) Phosphorylates NICD to make it a substrate for ubiquitylation and degradation
CBF1, Su(H) and LAG-1/Recombination signal binding protein for immunoglobulin kappa J region (CSL/RBP-J) Main canonical transcriptional co-factor for NICD
Cyclin C (CycC) Targets NICD for phosphorylation to make it a substrate for ubiquitylation and degradation
Dishevelled (Dsh/Dvl) Controls ligand-independent Notch trafficking; inhibits canonical Notch signalling
Deltex-1-4 (Dtx1-4) Controls Notch ubiquitylation, processing, and internalisation
Itchy, E3 ubiquitin protein ligase (Itch) Promotes ubiquitylation of NICD
Mastermind-like 1/2 (Maml1/2) Co-activator for NICD/CSL
Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-ϰb) NICD blocks NF-ϰb transcription of NF-ϰb target genes through binding to p50/cRel
NICD enhances NF-ϰb transcription of target genes by retaining NF-ϰb in the nucleus
Numb homolog (Numb) Suppresses Notch signaling by recruiting E3 ubiquitin ligases to ubiquitylate Notch
Controls Notch trafficking during asymmetric cell division
Smad family members (SMAD) Smads enhance Notch signalling; Notch fine-tunes signalling through Smads


Roles in Embryonic Development

The phylogenetically conserved Notch signalling pathway plays a crucial role in the development of multiple organ systems, and is a major regulator of stem cell fate. It is responsible for the regulation of the transcription of a number of signalling molecules, such as MyoD, Mash1 and GATA2, which are genes controlling the fate of myogenic, neurogenic and haematopoietic stem cells, respectively. [20] The following subsections will further elucidate the vital roles of Notch signalling during normal embryonic development. If you would like to better understand human embryonic development before reading this section, this page serves as a great resource.

Cardiovascular

If you would like to generally learn about and become familiar with cardiac development in the embryo before reading this section, click here!

Cardiomyocyte Specification and Differentiation

Expression during the appropriate window of the timeline of embryogenesis of Notch receptors, ligands and downstream effector molecules elucidates a role for the Notch pathway in the earliest stages of cardiac development. It has been found to restrict the expression of specific cardiogenic genes in a spatiotemporal manner and regulate cardiac field specification as early as during gastrulation. [21] Interestingly, Notch has been found to play both suppressive and promoting roles in cardiogenesis.

For example, it has been shown that Notch suppresses cardiomyocyte cell fate specification during early cardiogenesis. This has been demonstrated through studies such as that carried out by Rones and colleagues (2000), which used activation and inhibition of Notch signaling in Xenopus. [22]

On the other hand, studies such as that by Boni et al. (2008) have found that Notch signalling may also promote myogenesis from cardiac progenitor cells. [20] Cardiogenesis has also been promoted by downregulating Notch-1 activity in stem cells of embryos (Nemir et al., 2006).[23]

Despite the understanding that Notch signalling is crucial to embryonic myogenesis, the exact molecular mechanism remains elusive. Research by Buas and colleagues (2010) has explored such mechanisms by studying the Notch target, Hey1, which is known to suppress myogenic differentiation. They concluded that this inhibitory function of Hey1 is primarily mediated through binding near to myogenin and Mef2C promoters, which leads to cessation of target gene expression. [24]

Simplified Scheme of the Roles of Notch in Cardiac Development
Simplified Scheme of the Roles of Notch in Cardiac Development. Notch has been found to restrict the expression of specific cardiogenic genes in a spatiotemporal manner and regulate cardiac field specification early in development. Further after cardiac differentiation, Notch influences development of the AVC (atrioventricular canal), cardiac valves, ventricular trabeculae and the cardiac outflow tract. (This student-drawn image is based upon Figure 2 in the 2014 review by Zhou and Liu: Role of Notch signaling in the mammalian heart.[25])

Development of the Atrioventricular Canal

A study by Rutenberg et al. (2006) and another by Kokubo et al. (2007) implicate a role for Notch signaling in the region between the atria and the ventricles of the heart (the atrioventricular canal, or AVC).[26] [27] They used chicken and mouse models, respectively, to show that other signalling factors, Bmp2 and Tbx2, are restricted to the AVC region by Notch signalling during development.

Furthermore, Watanabe and colleagues (2006) showed that deletion of Notch targets increases Bmp2 expression and expansion of the AVC tissue, however other, non-Notch restrictive factors involved in AVC development are likely to exist.[28]

Heart Valve Development

In order for the heart valve to properly form in the embryo, endocardial to mesenchymal transformation (EMT) must occur. The Notch pathway, alongside Wnt and Bmp, has been found to regulate the process of EMT, defects in which can lead to congenital heart valve disease. Interestingly, Timmerman and colleagues (2004) demonstrated that this role of Notch may also promote oncogenic transformation. This team also showed that embryos exhibited abortive endocardial EMT if they were deficient in Notch signalling components in vivo and in vitro. [29]

A more recent paper by Wang et al. (2013) further explored the underlying signalling processes and interrelationships of molecules that impact EMT. They found that Jagged1-Notch1 signalling in cells of the endocardium potentiates expression of Wnt4, which in turn carries out paracrine action on adjacent AVC tissue to upregulate Bmp2 expression and thus signal EMT. [30]

Trabeculation

Trabeculation is the initial process of ventricular chamber development that forms a series of cardiomyocyte projections within the lumens of the ventricles of the heart (called trabeculae). Grego-Bessa and colleagues (2007) addressed the roles Notch plays in trabeculation through RBPJk (the gene product of which interacts with Notch) and Notch1 gene manipulation. Mutants of these two genes showed perturbed expression and signalling of EphrinB2, NRG1 and BMP10, alongside reduced proliferation of myocardiocytes. This research ultimately suggested that EphrinB2 is a direct target of Notch in the endocardium that simultaneously requires the Notch-dependent action of BMP10 and NRG1 in order for ventricular myocardium proliferation and differentiation to occur normally. [31] Furthermore, the important process of trabeculation has recently been shown to be controlled by sequential Notch activation by an investigation by D’Amato et al. (2015). [32]

Development of the Outflow Tract

About one third of congenital heart defects include malformations in the outflow tract which include the aorta, pulmonary arteries, aortic arch and ductus arteriosus. During cardiogenesis, neural crest cells interact with second heart field myocardium and endocardial mesenchyme. [33] Studies involving neurogenesis demonstrated the sensitivity of Notch signaling to temporal and spatial cues and showed the early Notch signaling initially controls the number of cells with a neurogenic fate and later dictates the lineage decision of neural versus glial cell fate. [34] Neural crest precursors for ideal patterning of the outflow tract are required in the Notch signaling pathway. The use of a dominant negative form of a master mind like protein (MAML) which has shown to be a pan Notch inhibitor in neural crest cells inactivates Notch signaling. This results in congenital heart defects like pulmonary artery stenosis and aortic arch patterning defects associated with defects in smooth muscle formation. [35] Part of the phenotype in defective neural crest cells is attributed to loss of Notch2 signalling as Notch2 inactivation in these cells produces small caliber aortas and pulmonary arteries because of defects in smooth muscle. [36] Jagged1 is the ligand involved which signals to Notch on neural crest cell surfaces to induce vascular smooth muscle cell differentiation. [37] Mutations in both JAGGED1 and NOTCH2 genes have been known to cause Alagille syndrome, further substantiating the importance of this pathway in proper formation of the outflow tract. [38]

Another study by Garg stresses the importance of Notch signaling in the outflow tract region. NOTCH1 mutations were seen in patients with aortic stenosis. Aortic stenosis results from the calcification of the aortic valve and is quite common in adults. However, in children, this may result in failure of the left ventricle to develop properly. NOTCH1 haploinsufficiency is also linked to early calcification and bicuspid aortic valve disease. This could be due to an early induction of Runx2 through the HRT genes. [39]



Central Nervous System

If you would like to generally learn about and become familiar with neural development in the embryo before reading this section, click here!

Simplified Diagram of Roles of Notch in Neuronal Differentiation
Simplified Diagram of Roles of Notch in Neuronal Differentiation. Notch influences the differentiation of embryonic stem cells into neural cells via signalling with RBP (recombining binding protein), cyclin D1 and Hes1. (This student-drawn image is based upon Figure 1 from Chuang, Tung and Lin's 2015 review: Neural differentiation from embryonic stem cells in vitro: An overview of the signaling pathways[40])

Early Neural Differentiation

Notch plays a major role in promoting neural commitment of cells. Lowell and colleagues (2011) used genetic manipulation to discover that the phenotype of stem cells is not affected by constitutively activated Notch in mouse embryonic stem cells (mESCs), however, interfering with Notch signalling -for example by pharmacological means- did impede neural fate determination. This role required Notch signalling via fibroblast growth factor (FGF) receptors. Furthermore, the conservation of the Notch signalling pathway within pluripotent stem cells is implied due to the existence of Notch ligands in stromal cells in human embryonic stem cells (hESCs) that induce neural differentiation. [41]

Das and colleagues (2010) manipulated Notch protein levels during specific stages of neural differentiation and found that if Notch signalling pathways were activated during day 3 of neural development for 6 hours, cell proliferation was dramatically enhanced. This was attributed to the induction by Notch of cyclin D1 expression. Without Notch signalling during neural development, there was reduced cyclin D1 levels. Manipulation of mESCs to express a dominant negative form of cyclin D1 resulted in abrogation of cell proliferation stimulated by Notch. Overall these results imply a temporally-specific role for Notch in CNS development, and that it requires cyclin D1 as a signalling molecule.[42]

Hes1 has also been shown to play a Notch-mediated role in neural progenitor maintenance, which is essential for proper development since cells need to proliferate only during specific periods in the embryo for the correct number, cell type and function to result. Shimojo, Ohtsuka and Kageyama (2008) used real time imaging in mice to show that Notch induces oscillatory expression of Hes1 and other Notch target genes to maintain the complex temporal organisation of events in neural development. They concluded that further research could elucidate more about Hes1 oscillations in regards to neural progenitor maintenance, proliferation and differentiation [43].

Lastly, as well as influencing neural progenitor differentiation, it has been found that Notch also impacts the development and maintenance of polarised neural structures in the embryo. This was concluded from a knockout study in mice that elucidated a role for RBP (recombining binding protein), downstream of Notch, in modulating neural differentiation and maintaining rosette structure (an experimental in vitro correlate used for neural tube development) [44]. The researchers concluded that RBP knockouts lacked canonical Notch signalling and as a result showed multiple neurulation defects.

Other Systems

As aforementioned, Notch plays a wide array of roles in embryonic development, the follow table summarises these roles by each organ system:

Summary Table of Examples of Notch Signalling in Developmental Processes (adapted from Table 1[19])

Organ/Tissue Processes regulated by Notch
Brain Controls the balance between gliogenesis and neurogenesis; stem cell maintenance[45][46]; organisation and maintenance of polarity during early development[44]
Craniofacial structures Palate morphogenesis: loss of Notch signalling results in cleft palate, fusion of the tongue with the palatal shelves, and other craniofacial defects; also involved in tooth development
Ear Defines the presumptive sensory epithelium; determines hair cell and supporting cell fates
Esophagus Regulates esophageal epithelial homeostasis
Heart Cardiac patterning, cardiomyocyte differentiation, valve development, ventricular trabeculation, outflow tract development
Intestine Controls proliferation and differentiation, including absorptive vs. secretory cell fates
Limbs Apical ectodermal ridge (AER) formation and digit morphogenesis, especially regulation of apoptosis
Lungs Lateral inhibition between tracheal cells prevents extra cells from assuming the lead position during tracheal branching morphogenesis
Neural crest Controls patterning of neural crest precursors for the outflow tract region of the heart; regulates the transition from Schwann cell precursor to Schwann cell, controls Schwann cell proliferation and inhibits myelination; controls melanocyte stem cell maintenance
Pancreas Specifies endocrine cell differentiation through lateral inhibition: endocrine lineage cells inhibit endocrine differentiation of their neighboring cells; maintains pancreatic endocrine precursor cells, inhibits terminal acinar cell differentiation; controls pancreatic epithelium branching and bud size
Skin Regulates cell adhesion, control of proliferation, hair follicle or feather papillae differentiation and homeostasis
Thyroid Regulates the numbers of thyrocyte and C-cell progenitors and regulates differentiation and endocrine function of thyrocytes and C-cells[47]
Vasculature Regulates arteriovenous specification and differentiation in endothelial cells and vascular smooth muscle cells; regulates blood vessel sprouting and branching

Quiz Your Notch-Knowledge on Embryonic Development!

In the development of which organ does Notch exert lateral inhibition to differentiate endocrine cells?

  The heart
  The central nervous system
  The skin
  The pancreas


Roles in Animal Development

The Notch signalling pathway doesn't just play a significant role in human development, but also affects events such as neurogenesis and myogenesis in some animals (thus why these animals are often used as models for human development in research!). Read below for information on some of these roles for Notch.

Animals Whose Developmental Processes are Affected by Notch.
Animals Whose Developmental Processes are Affected by Notch. This is a simplified representation of a few examples of the animals (Drosophila flies[48], C. elegans[49], and the zebrafish[50]) in which development is influenced by the Notch signalling pathway, and their scientific names.

Drosophila melanogaster

(The Fly)

Unlike the four Notch paralogs in humans, only one Notch homolog is present in Drosophila.[14]

Both canonical and non-canonical Notch signalling are involved in the structural and physiological responses and functional plasticity of olfactory receptor neurons in reaction to prolonged odour exposure.[51][52] Research has also shown that Notch is crucial for the formation of longitudinal connections in the Drosophila CNS. Kuzina, Song, and Giniger (2011) created temperature-sensitive mutations of Notch genes that prevented the development of mature longitudinal axon tracts. Additionally, it was found that the Notch phenotype appears at the earliest stages of the development of longitudinal connections in the CNS by observing early stage 13 embryos.[48]

Caenorhabditis elegans

(The Worm)

The Notch pathway in C. elegans occurs throughout development in populations of equipotent cells for neuronal function in postmitotic differentiated neurons. In these postmitotic neurons, there is a specialised post-embryonic development stage knows as 'dauer'. The Notch pathway is activated when cell signalling downstream of the developmental decision enter dauer. The Notch receptor glp-1 and the ligand lag-2 are expressed in the dauer stage and aid in maintaining this stage. Another Notch receptor, lin-12, functions upstream of insulin signalling components to promote conditions for growth and enhance dauer recovery. [49]

Danio rerio

(The Zebrafish)

The expression of Notch in the myogenic region and apical ectodermal ridge (AER) of the developing zebrafish fin is similar to what has been observed in developing chicken and mouse limbs, which shows how highly conserved the role of Notch signalling is in the development of appendages in vertebrates. During zebrafish embryogenesis, the timing and positioning of fin formation are dependent on Notch signalling. Notch is also involved in the actual processes of fin formation, such as AER signalling, chondrogenic differentiation, and myogenesis. In zebrafish embryos with defective Notch signalling, it was observed that the skeletal muscle fibres were thin and fragmented and the structure of the sarcomeres was significantly compromised. [50]

Expand this for an image from this research study
Abnormal pectoral fins are formed in Notch signalling disrupted larvae
Abnormal pectoral fins are formed in Notch signalling disrupted larvae. (A–F) Live pictures of 5 dpf larvae. (A’) A pectoral fin from a sibling embryo showing the cartilaginous endoskeletal disc with individualized cells surrounded by thin matrix deposits, the fin fold and the chleitrum (n = 17) (E, E’). A similar pectoral fin was found in a DMSO-treated embryo (n = 9). Pectoral fins of Notch signalling disrupted embryos such as mibta52b (n = 18) (B, B’), jagged2 (n = 10) (C, C’) and Su(H)1+2 (n = 12) (D, D’) morphants and DAPT-treated embryos (n = 10) (F, F’) showing disorganized endoskeletal disc cells. cl, chleitrum; ed, endoskeletal disc; ff, fin fold.[50]



Quiz Your Notch-Knowledge on Animal Development!

In which animal (as described in this section) has it been shown that defective notch signalling leads to thinning and fragmentation of skeletal muscle fibres?

  Drosophila melanogaster
  I don't know!
  Caenorhabditis elegans
  Danio rerio


Summary of Functions of Proteins Involved in Notch Signalling (adapted from Table 1[53])

Mammals Drosophila C.elegans Function
Notch 1-4 Notch Lin-12, Glp-1 Single transmembrane receptor and transcription factor
Delta 1, Delta3-4, Jagged1-2 Delta, Serrate APX-1, LAG-2, ARG-1, DSL-1 Single transmembrane ligands of the Notch receptor
CBF1/RBPJK
Mastermind1-3
Su(H)
Mastermind
Lag-1
Lag-3
DNA-binding transcription factor
Transcriptional co-activator
Lunatic, manic, and radical Fringe Fringe Modifies both Notch and its ligands
ADAM10, ADAM17 Kuzbanian, Kuzbanian-like, TACE SUP-17, ADM-4 Metalloproteases targeting S2 Notch cleavage sites
Presenilin 1-2, nicastrin, APH1, PEN2 Presenilin, nicastrin, APH1, PEN2 SEL-12, APH-1, APH-2, PEN2 Proteins of the γ-secretase complex, which targets Notch S3 and S4 cleavage sites
Mind bomb, skeletrophin, neuralized 1-2 Mind bomb 1-2, neuralized Y47D3A.22 E3 ubiquitin-protein ligases that targets Delta and Jagged/Serrate and regulate their endocytosis


Abnormalities in Notch Signalling

Mutations in Notch genes and hence defects in Notch signalling have been implicated in the pathogenesis of several inherited diseases in humans.[54][55] The diseases listed in this summary table are only a few examples and are described in more detail below.

Disease Gene Phenotype
Alagille syndrome (OMIM118450, OMIM610205) JAG1, NOTCH2 Developmental abnormalities of the heart, liver, eye, and skeleton. Neonatal jaundice and cholestasis are early symptoms.
CADASIL (OMIM125310) NOTCH3 Autosomal vascular disorders linked to ischemic strokes, dementia, and premature death.
T-cell acute lymphoblastic leukaemia (OMIM613065) NOTCH1 (mutation in heterodimerization domain or PEST domain) Tumour derived from T-cell progenitors. Symptoms include anaemia and enlargement of lymph nodes in the liver and/or spleen.
Spondylocostal dysostosis (OMIM122600) DLL3, Lunatic fringe Vertebral segmentation defects as well as rib abnormalities.

Alagille Syndrome

Alagille syndrome (AGS) is an autosomal dominant, multisystem disorder that mainly affects the liver, heart, and kidney. Diagnostic characteristics of the disease include liver disease, cardiac disease, vertebral defects, eye conditions, and facial features, as well as renal and vascular abnormalities.[56] Two variants of AGS, type 1 and type 2, have been identified, and each is related to different defects in the Notch pathway. In clinically diagnosed cases of AGS type 1 (which accounts for approximately 97% of all cases of AGS), a mutation in the gene encoding the Notch ligand Jagged1 (Jag1) has been identified as a contributing factor.[57] In the much rarer AGS type 2, a mutation in the NOTCH2 gene has been implicated in the manifestation of AGS. The mechanisms by which mutations in JAG1 and NOTCH2 lead to pathogenesis of AGS are not well understood, but it is known that normal Notch signalling is important in angiogenesis. Therefore, it is thought that abnormal JAG1 or NOTCH2 will cause disruptions to the pathway that leads to the vascular disorders present in AGS. This is also evidenced by research that JAG1 knockout mice suffer premature death due to vascular defects.[58] It is possible that AGS caused by a mutation in NOTCH2 alone will lead to a different presentation than AGS patients with mutated JAG1; however, this is still undergoing further research. It is also unclear why AGS type 2 occurs at a significantly lower incidence than AGS type 1.[59]

Spondylocostal Dysostosis

Spondylocostal Dysostosis (SD) is a collective term for conditions characterised by anomalies relating to rib and spine. The vertebrae are fused and shaped abnormally which can result in scoliosis. Similarly, the ribs may also be fused together or in some cases completely missing. As a result, people with this condition have short trunk dwarfism where their bodies are short in stature but have normal length limbs. Genetic changes are known to cause SD. SD Type 1 is the most prevalent form of this disease which is caused by the mutation in the delta like canonical Notch ligand 3 (DLL3 gene).[60] DLL3 provides the information for making a protein that regulates the Notch signalling pathway. The DLL3 protein in conjunction with the Notch pathway is primarily responsible for preventing the fusion of future vertebrae in a process known as somite segmentation.[61] An interruption in the Notch pathway inhibits the somite segmentation resulting in abnormalities relating to the ribs and spine as seen in SD. 25 percent of SD arise from mutations in the identified genes and further research suggest that other genes involved in the Notch signalling pathway may also be related to SD.

Cerebral Autosomal-Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL)

CADASIL is an autosomal-dominant disease of the small to medium-sized arteries, mainly in the brain, that leads to dementia and disability in mid-life. The symptoms, age of onset, and prognosis are variable. Distinguishing symptoms include subcortical ischemic events (60-80% of cases), cognitive impairment (60% of late stage disease), migraines (30-40%), mood disturbances (30%), and apathy (40%).[62][63] Approximately 5-10% of CADASIL patients also experience seizures. The mean age of onset is 45 years of age and the disease duration is between 10–40 years. More than 95% of CADASIL cases present with pathogenic mutations in NOTCH3 (located on chromosome 19p13), specifically the epidermal growth factor-like repeat domain.[63][64] The NOTCH3 gene is involved in the normal development of blood vessels in both fetal and adult brains. In adults, NOTCH3 is expressed in the smooth muscle cells of arteries.[62] There is currently no effective treatment available for CADASIL.

Congenital Heart Defects

Aortic Valve Disease

Loss of NOTCH1 expression in proximity to calcific nodules in human aortic valves
Loss of NOTCH1 expression in proximity to calcific nodules in human aortic valves. (A) Representative sections from control (A,B) and diseased (C-F) aortic valve cusps. (B) is high magnification image of boxed area in (A) and (D,E) are higher magnification of region in (C) while image in (F) shows another calcified aortic valve. Expression of Notch1 intracellular domain (NICD) is found in the thickened fibrosa of diseased aortic valve (C,E) as compared to the acellular fibrosa of control valves (A,B). However, there is significant loss of NICD expression in cells residing adjacent to calcific nodules (D,F). The fibrosa is oriented upward in all panels, and scale bars equal 100 microns (B,D,E,F are at same magnification). Brown signal represents NICD expression while nuclei are counterstained in blue.[65]

Calcification of the aortic valve is a leading cause of adult heart disease. Mutations in NOTCH1 have been found to cause various aortic valve abnormalities, including the development of a bicuspid aortic valve (as opposed to tricuspid) and valve calcification.

  A study by Garg et al. (2005) looked at mouse embryos and the expression of Notch1 during development. It was found that during normal embryonic development, Notch1 was abundantly expressed in the outflow tract mesenchyme (which develops into the heart valves) and the endocardium, as well as in the endothelial layer and mesenchyme of the aortic valve leaflets at the time of arterial trunk septation. Abnormal Notch1 led to the death of mice from vascular endothelial defects. Garg et al. then investigated whether NOTCH1 was involved in calcium deposition and therefore valve calcification. This is thought to be due to differentiation of valve cells into osteoblast-like cells. This leads to the upregulation of osteopontin, osteocalcin, and other osteoblast-specific genes, which is normally regulated by the transcription factor Runx2. Runx2 is known to be upregulated in animal models of valve calcification. Garg et al. found that Notch1 was capable of repressing Runx2 activation. Heart tissue and vasculature are normally abundant with the hairy-related transcriptional repressors Hrt1 and Hrt2 (also called Hey1 and Hey2), which are normally activated by Notch and are key mediators of the Notch pathway. Hrt1 and Hrt2 were both expressed in the endothelium of the mice aortic valve leaflets, the endocardium, and the vascular endothelium. They were found to inhibit the activation of osteoblast-specific genes by Runx2. It was thought that Hrt proteins can repress Runx2 by physical interaction as well as mediate Notch1 repression of Runx2. Therefore, a defect in Notch will cause upregulation of Runx2, due to the lack of repressor activity by Notch itself and the activation of the repressors Hrt1 and Hrt2. This leads to the expression of osteoblast genes that results in the differentiation of valvular cells into osteoblast-like cells and ultimately causes aortic valve calficiation.[66]

The picture to the right is a histological image of aortic valve disease from a study performed in rats investigating molecular changes that occurred when Notch signalling was suppressed in the aortic valve.[65]

Tumourigenesis

Notch has been shown to be involved in cancer development and can act as both an oncogene and a tumour suppressor gene.[67][54] Defective Notch signalling has been implicated in the pathogenesis of several human cancers, such as acute myelogenous leukemia (overexpression of Jagged1 and Notch1), multiple myeloma (overexpression of Jagged1/2 and Notch1/2), and B-cell–derived Hodgkin lymphoma (overexpression of Jagged1 and Notch1).[68]

Elevated levels of Notch ligand proteins and/or mRNA have been observed in several cancers. Upregulated Jagged1 mRNA has been seen in human pancreatic cancer; Jagged1 protein overexpression has also been studied in prostate, cervix, and brain cancers. Cervical cancers have shown upregulated Jagged2 mRNA. Additionally, elevated Dll1 mRNA has been observed in cervical cancers, as well as in human brain cancers at both Dll1 mRNA and protein levels. Additionally, defective Notch receptor expression has been implicated in cancer as well. For example, elevated Notch1 protein expression has been found in cervical, colon, lung, pancreas, skin, and brain cancers. Protein overexpression of Notch3 and Notch4 has also been seen in malignant melanoma and pancreatic cancer.[68]

Interestingly, research has shown that Notch signalling can also have a tumour-suppressive effect. Studies have indicated that Notch signalling can inhibit proliferation and even stimulate apoptosis in malignant B-cells. Furthermore, the Notch1 intracellular domain has also shown the ability to induce growth arrest and apoptosis in Hodgkin lymphoma and multiple myeloma cells.[68]

T-Cell Acute Lymphoblastic Leukaemia
T-cell acute lymphoblastic leukaemia (T-ALL) is an example of how abnormal Notch signalling can lead to the development of cancer. T-ALL is an aggressive childhood cancer of the immune system's T-cells. The NOTCH1 gene encodes the Notch1 receptor, which is involved in the determination of pluripotent cell progenitors to T-cells and the organisation of these cells in the developing thymus. Activating mutations in the extracellular domain and/or the C-terminal PEST domain of Notch1 have been identified in more than half of all cases of T-ALL.[69]

Other Examples of Roles of Notch in Cancer (adapted from Notch in disease[54])
Gene Role of Notch Disease
NOTCH1
NOTCH2
Oncogene
Tumour suppressor
Pancreatic ductal adenocarcinoma
NCSTN
MAML1
APH1A
NOTCH2
Tumour suppressor Chronic myelomonocytic leukaemia
NOTCH1 Oncogene Chronic lymphocytic leukaemia
Activated NOTCH Tumour suppressor Hepatocellular carcinoma
NOTCH1
NOTCH2
NOTCH3
Tumour suppressor Head and neck squamous cell carcinoma
NOTCH Tumour suppressor B-cell acute lymphoblastic leukaemia
NOTCH1 Oncogene Non-small-cell lung cancer
NOTCH1 Oncogene T-cell acute lymphoblastic leukaemia


Quiz Your Notch-Knowledge on Abnormalities in Notch Signalling!

What is the name for the collection of rib and spine abnormalities that result from interruption in the Notch pathway that leads to inhibition of somite segmentation in the embryo?

  Alagille Syndrome
  T-cell acute lymphoblastic leukaemia
  Spondylocostal Dysostosis
  CADASIL


Current Areas of Research and Future Directions

Notch has been identified as having a role in the development of several cancers, and therefore research has recently begun investigating the use of Notch inhibitors in cancer treatment.[70] γ-secretase inhibitors (GSIs), which inhibit the normal cleavage of Notch at the cell membrane and hence NICD release, have been a popular area of research. In the specific case of desmoid tumours, it was observed that treatment GSIs resulted in decreases in NICD and Hes1 expression in the tumour cells. Overall, tumour cell migration and invasion were decreased and cell growth was also inhibited.[71] GSIs have also been shown to have an inhibitory growth effect on pancreatic, breast, and lung cancers, but unfortunately they also produce side effects in vivo.[72] Therefore continued study into GSIs is required to make them safe for human treatment. Ultimately, with ongoing research, it is possible that the Notch pathway will eventually become an effective therapeutic target for cancers.

To search and read recent PubMed research and review articles on Notch, click here!

Want to Read More About Notch?

The OMIM (Online Mendelian Inheritance in Man) database contains extensive information about the Notch genes and associated proteins and is a great place to go if you want to find out more about the Notch pathway in humans! Click on these links to learn more about the Notch genes that encode the human receptors and ligands:

  • NOTCH1 | NOTCH2 | NOTCH3 | NOTCH4
  • JAG1 | JAG2
  • DLL1 | DLL3 | DLL4

    You can also look at these papers for examples of reviews and research papers that reinforce the diversity of how the Notch pathway functions in organisms.
    Notch influences adult development as well!
    Interestingly, Notch does not only play a role in human embryonic development, but has also been found to influence developmental processes after birth. One of these processes is neurogenesis, for example.

    Not(ch) just development: Notch signalling in the adult brain.

    This review explains that the Notch signalling pathway, which is so often regarded as a major molecular player in development, actually has important functions related to neural cells in the adult. As you would have read in the Abnormalities in Notch Signalling section, Notch abnormalities are involved in a number of diseases, which underlines its importance in maintaining adult brain function. Some highlights of this review are: Figure 2 provides a schematic representation of precise roles Notch activation can play in the adult brain; explanations of the ways that Notch influences brain plasticity; and the discussion of conflicting results of studies exploring the role of Notch in stroke recovery.[73]

    Notch modulation for therapy
    As you would have read in the Abnormalities in Notch Signalling section, Notch can play different roles in cancer development. An interesting review further elucidates that modulation of Notch signalling is actually being investigated as a potential target for cancer therapy:

    Targeting the Notch signaling pathway in cancer therapeutics

    This articles focuses on reviewing data emerging from recent research, explaining how Notch contributes to the development of a number of cancers, and then outlining the current focuses of investigations into how to therapeutically manipulate the Notch pathway. Some highlights from this review are: the detailed description of how Notch interacts with other signalling pathways such as Wnt and the signalling cytokine Interleukin-6; explanations of the seemingly conflicting roles of Notch as a tumour promoter and suppressor; and discussion of the findings of various Notch-related studies in cancer stem cells.[74]

    Notch influences reproduction in worker honey bees!
    In our Roles in Animal Development section we discussed Notch in relation to a number of animals, however one that we did not cover was the worker honey bee, or Apis mellifera. A recent study shows that Notch also plays a role in these honeybees!

    Notch signalling mediates reproductive constraint in the adult worker honeybee.

    This research paper essentially concludes that in the worker honeybee Notch signalling constrains reproduction between males and females; they were the first team to elucidate a molecular mechanism underlying the link between adult ovary activity in bees and the presence of the queen bee. Some highlights from this unique and interesting study are: the background explanation that female worker bees have suppressed activity due to pheromones produced by the queen; the finding that Notch plays a part in this inactivation of reproductivity; and the discussion about social control of reproduction and evolution.[75]

    Notch signalling is involved in embryo implantation!
    Notch is essential for many developmental embryonic processes, as described in our Roles in Embryonic Development section. Interestingly, research has shown that Notch signalling also has an important role in the initial process of establishing a successful pregnancy. Specifically, normal Notch signalling has been seen to be a critical factor in fetal-maternal communication during implantation and placentation. (To read more about these processes, go to these pages on Weeks 1-2 and Week 3 of development.)

    Fetal-maternal communication: the role of Notch signalling in embryo implantation.[76]

    This review describes both human and animal studies of Notch signalling during pregnancy. It explains research of Notch signalling at the human blastocyst-maternal interface, and that Notch signalling has been observed in both the maternal endometrium and the blastocyst. Furthermore, Notch signalling is also important for the processes of implantation and placentation. Overall, the review summarises findings of Notch signalling in endometrial-trophectoderm interactions during the implantation, regulation of extravillous trophoblast invasion and spiral artery remodelling in the decidua, and angiogenesis in the placenta, as well as how abnormal Notch signalling is related with impaired placentation and pre-eclampsia.


    Glossary

    AER 'Apical Ectodermal Ridge' is a structure that forms from the ectodermal cells at the distal end of each limb bud. The AER acts as a major signalling centre in order to ensure proper limb development.
    Apoptosis Programmed cell death which occurs in the development of many systems.
    Calcification The accumulation of calcium salts in body tissue. It normally occurs in the formation of bone, but abnormally calcium can be deposited in soft tissue causing it to harden.
    Cardiogenic From cardiogenesis - meaning formation of the heart.
    Cardiomyocyte The muscle cells (myocytes) that make up the cardiac muscle
    Congenital (Of a disease or physical abnormality) present from birth.
    Craniofacial Meaning relating to the cranium and the face.
    Cyclin D1 A protein required for progression through the G1 phase of the cell cycle. Read more about it here, or see information on its encoding gene here: OMIM168461.
    Endocardial From endocardium - meaning the thin, smooth membrane which lines the inside of the chambers of the heart and forms the surface of the valves.
    Endocrine As in the endocrine system, refers to glands that secrete hormones or other substances directly into the blood.
    Epithelium The thin tissue that forms the outer layer of a body's surface and also lines the alimentary canal and other hollow structures.
    Gastrulation Describes the formative process of the formation of the trilaminar embryo containing the three germ layers.
    Gliogenesis The formation and development of glial cells (astrocytes, oligodendrocytes, Schwann cells, microglia).
    Haematopoietic From haematopoiesis - meaning the formation of blood cellular components
    Haploinsufficiency When a diploid organism has only a single functional copy of a gene (with the other copy inactivated by mutation), and so the total level of a gene product (a particular protein) produced by the cell is about half of the normal level.
    Homeostasis The ability to maintain a constant internal environment in response to environmental changes.
    Ligand A molecule that binds to a specific binding site on a protein (receptor).
    Mef2C promoters Myocyte-specific enhancer factor 2C is a transcription factor in the Mef2 family that is encoded by the gene MEF2C in humans (OMIM 600662). This gene is involved in cardiac morphogenesis and myogenesis, as well as vascular development. It is thought to also possibly play a role in neurogenesis.
    Mesenchymal From mesenchyme - meaning a loosely organised, mainly mesodermal embryonic tissue which develops into connective and skeletal tissues, including blood and lymph.
    Morphogenesis Refers to the origin and development of morphological characteristics.
    Myogenic From myogenesis - meaning formation of muscle.
    Myogenin Also known as myogenic factor 4, this is a muscle-specific basic-helix-loop-helix transcription factor involved in the coordination of skeletal muscle development or myogenesis and repair.
    Neural Crest In vertebrate embryos, this is a transient structure that gives rise to most of the peripheral nervous system and to a number of non-neural cell types such as the smooth muscle cells of the cardiovascular system.
    Neurogenic From neurogenesis - meaning the formation of nervous tissue.
    Neurulation The folding process in vertebrate embryos whereby the neural plate transforms into the neural tube.
    NICD Notch intracellular domain
    Oncogenic From oncogenesis, also known as tumorigenesis or carcinogenesis - meaning the formation of a cancer, whereby normal cells are transformed into cancer cells.
    Paracrine A hormone that exerts an effect only in the vicinity of the gland secreting it.
    Protease An enzyme which breaks down proteins and peptides.
    Runx2 Runt-related transcription factor 2 is a protein that in humans is encoded by the RUNX2 gene (OMIM600211). RUNX2 is an important transcription factor associated with osteoblast (bone substance-secreting cells) differentiation.
    Stromal cells The connective tissue cells of any organ. They support the function of the parenchymal cells of that organ. Fibroblasts and pericytes are among the most common types of stromal cells.

    Further Glossary Links

    A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | #

    References

    1. 1.0 1.1 Moore, K.L., Persaud, T.V.N. & Torchia, M.G. (2015). The developing human: clinically oriented embryology (10th ed.). Philadelphia: Saunders.
    2. 2.0 2.1 As reviewed by <pubmed>10075488</pubmed>
    3. 3.0 3.1 As reviewed by <pubmed>19255248</pubmed>
    4. Dexter, J. (1914). The Analysis of a Case of Continuous Variation in Drosophila by a Study of Its Linkage Relations. The American Naturalist, 48(576), 712-758. Retrieved from http://www.jstor.org/stable/2455888
    5. Morgan, T. H. (1917). The theory of the gene. The American Naturalist, 51(609), 513-544.
    6. <pubmed>16588136</pubmed>
    7. <pubmed>13635554</pubmed>
    8. <pubmed>6403942</pubmed>
    9. <pubmed>3097517</pubmed>
    10. <pubmed>2338245</pubmed>
    11. <pubmed>22558462</pubmed>
    12. <pubmed>25083094</pubmed>
    13. <pubmed>27404588</pubmed>
    14. 14.0 14.1 14.2 14.3 14.4 Yamamoto, S., Schulze, K.L. & Bellen, H.J. (2014). Introduction to Notch Signalling. Notch Signaling: Methods and Protocols. Methods in Molecular Biology: 1187
    15. 15.0 15.1 As reviewed by <pubmed>22397947</pubmed>
    16. <pubmed>27040987</pubmed>
    17. 17.0 17.1 As reviewed by <pubmed>9892565</pubmed>
    18. <pubmed>9247339</pubmed>
    19. 19.0 19.1 As reviewed by <pubmed>21828089</pubmed>
    20. 20.0 20.1 <pubmed>18832173</pubmed>
    21. <pubmed>19580804</pubmed>
    22. <pubmed>10934030</pubmed>
    23. <pubmed>16690879</pubmed>
    24. <pubmed>19917614</pubmed>
    25. <pubmed>24345875</pubmed>
    26. <pubmed>17021042</pubmed>
    27. <pubmed>17259303</pubmed>
    28. <pubmed>16554359</pubmed>
    29. <pubmed>14701881</pubmed>
    30. <pubmed>23560082</pubmed>
    31. <pubmed>17336907</pubmed>
    32. <pubmed>26641715</pubmed>
    33. <pubmed>20201902</pubmed>
    34. <pubmed>16429119</pubmed>
    35. <pubmed> 17273555 </pubmed>
    36. <pubmed> 18330927 </pubmed>
    37. <pubmed> 18245384 </pubmed>
    38. <pubmed> 16575836 </pubmed>
    39. <pubmed> 16025100 </pubmed>
    40. <pubmed>25815127</pubmed>
    41. <pubmed>16594731</pubmed>
    42. <pubmed>20887720</pubmed>
    43. <pubmed>18400163</pubmed>
    44. 44.0 44.1 <pubmed>23675446</pubmed>
    45. <pubmed>21262462</pubmed>
    46. As reviewed by <pubmed>20816397</pubmed>
    47. <pubmed>21364918</pubmed>
    48. 48.0 48.1 <pubmed>21447553</pubmed>
    49. 49.0 49.1 <pubmed>18599512</pubmed>
    50. 50.0 50.1 50.2 <pubmed>23840804</pubmed>
    51. <pubmed>26986723</pubmed>
    52. <pubmed>26011623</pubmed>
    53. As reviewed by <pubmed>17761886</pubmed>
    54. 54.0 54.1 54.2 As reviewed by <pubmed>23729744</pubmed>
    55. As reviewed by <pubmed>22306179</pubmed>
    56. As reviewed by <pubmed>26548814</pubmed>
    57. As reviewed by <pubmed>11745040</pubmed>
    58. As reviewed by <pubmed>21934706</pubmed>
    59. <pubmed>16773578</pubmed>
    60. <pubmed>10742114</pubmed>
    61. <pubmed>11236715</pubmed>
    62. 62.0 62.1 <pubmed>21045164</pubmed>
    63. 63.0 63.1 <pubmed>20301673</pubmed>
    64. <pubmed>24579972</pubmed>
    65. 65.0 65.1 <pubmed>22110751</pubmed>
    66. <pubmed>16025100</pubmed>
    67. As reviewed by <pubmed>14570040</pubmed>
    68. 68.0 68.1 68.2 As reviewed by <pubmed>16291593</pubmed>
    69. <pubmed>15472075</pubmed>
    70. As reviewed by <pubmed>27732970</pubmed>
    71. <pubmed>26349011</pubmed>
    72. As reviewed by <pubmed>27688721</pubmed>
    73. As reviewed by <pubmed>21505516</pubmed>
    74. <pubmed>26767041</pubmed>
    75. <pubmed>27485026</pubmed>
    76. As reviewed by <pubmed>24357662</pubmed>