Signal Transduction

Notch in the maintenance of an intestinal stem cell compartment

Activation of Notch causes potent inhibition of differentiation in a number of developmental contexts, and it has been associated with amplification of somatic stem cells, such as the neural and haematopoietic stem cells.63,64 The intestine too has its stem-cell compartments, present in the crypts (see Figure 14.14 page 434). As described in Chapter14, Wnt signalling plays an

important role in driving proliferation of these stem cells (as well as their early progenitors). In the case of the small intestine, they do not differentiate until they are pushed out of the crypt and start moving up the walls of the villi. Once they reach the top, they are shed. Here we indicate how Notch acts in concert with Wnt by preventing differentiation both of the stem cells and

of their early progenitors (Figure 22.14). Moreover, Notch signalling is vital to assure a balanced mixture of secretory and absorptive cells.

The role of Notch in the development of intestinal crypts is well indicated by the finding that the intestines of zebrafish that are DeltaD (ligand) mutants have an increased number of secretory goblet cells, at the expense of absorptive epithelial cells (enterocytes).65 The default for a crypt progenitor cell is differentiation towards goblet cells. To generate a balanced mixture of absorptive and secretory cells, lateral inhibition, mediated by Delta–Notch signalling, is needed. This is also true for mice in which multiple Notch pathway components are expressed. Mice lacking hes1 have few if any absorptive cells and reveal an excess of secretory and enteroendocrine cells. Induction of a constitutively active form of Notch1 in all the cells of the intestinal epithelium causes a loss of secretory goblet and a reduced number of enteroendocrine cells. Instead, there is an expansion of immature absorptive cells, some of which still divide far beyond their crypts.66

Adenomas, in which there is a highly active Wnt pathway, due to loss of function of the adenomatous polyposis coli (APC) protein, also employ the Notch pathway to suppress differentiation. They contain all necessary

signalling components and express the Hes1 effector gene. Strikingly, when APC-deficient mice carrying numerous polyps were fed an inhibitor of-secretase, a large number of crypts regained a more or less normal histology, populated by numerous differentiated goblet cells.67,68

Conversely, Notch is required for differentiation of stem cells as well as the production of an appropriate fraction of enteroendocrine cells in the stem cell compartment of the midgut in Drosophila.69

Cross-talk with other signal transduction pathways

We end this chapter with examples that show that Notch interacts with other signal transduction pathways (or the other way round), and that this interaction occurs at different levels.

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FIG 22.14  Notch regulates the size of the intestinal stem cell compartment and determines cell lineage of progenitor cells.

The intestinal crypt, which harbours the stem cell compartment, is under the influence of both Wnt and Notch. Notch prevents differentiation of the stem cell and its early progenitors. It also plays a role, possibly through lateral inhibition, in the lineage decision of the later-stage progenitor cells. Thus, receiving cells maintain an enterocyte (absorptive) phenotype whereas the sending cell turns into either a goblet or a Paneth cell (secretory cell). Loss of Notch therefore gives rise to a minute stem cell compartment and most of the progenitors turn into goblet or Paneth cells. Conversely, an excess of Notch prevents differentiation into secretory cells and leads to an excess of absorptive cells that continue to divide beyond the crypt region.

Cross-talk at the level of the membrane

Notch interacts with the Wnt pathway in Drosophila70 and at least two pathway components are implicated. It binds Dishevelled, an effector protein immediately downstream of the Wnt receptor Frizzled (Fz), and it binds Armadillo ( -catenin), the component of the Wnt pathway that relays the signal into the nucleus (see Chapter 14, in particular Figure 14.8, page 429). Both interactions occur over a broad region of the intracellular domain of Notch. Gain-of-function mutants of Notch antagonize Wnt signalling. The observation that both Dishevelled and Armadillo interact directly with Notch suggests that sequestration of essential signalling components forms the basis of this antagonism. Notch does not blunt the action of Wnt, but it may constitute an important buffer that raises the threshold level for Wnt

signalling (Figure 22.15). In other words, Notch acts as a filter that cuts off weak signals (noise) and this could serve to create sharp boundaries between responding and non-responding cells in developing embryos.71,72

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Cross-talk at the level of gene expression

Notch cooperates with the TGF signal pathway at the level of induction of gene expression75 (Figure 22.16). In myoblast and adult neural stem cells, where addition of Notch and TGF prevent differentiation, Nicd and Smad3/Smad4 synergize by forming a protein complex that interacts with CSL. This leads to a higher level of expression of Hes1 than either of the pathways alone can achieve. Hes1 protein, in turn, prevents expression of myogenic factors such as the bHLH protein MyoD.76

A similar synergy at the level of transcription occurs between BMP4 and Notch, but the context is different. Here we deal with vascular endothelial cells that show enhanced motility under the influence of BMP4 (member of the TGF family of cytokines, see Chapter 20). However, the motility of cultured cells ceases when the density rises to a level where frequent cellcell contacts occur. During contact, Delta or Jagged1 binds and activates Notch. The subsequently liberated Nicd associates with BMP4-activated

Smad1/Smad4 complexes and with CSL. Together they induce high levels of expression of the bHLH protein Hey1 which, in turn, causes destruction of Id1 (Figure 22.16).73 Expression of Id1, also a bHLH, which sequesters transcription factors away from their favoured promoter elements, is apparently essential for maintaining the motility of endothelial cells. It is known for its role in progression of the G1 phase of the cell cycle. In the context of migration, it suppresses expression of thrombospondin-1, a component of the extracellular matrix that acts to prevent new blood vessel formation (angiogenesis).

Cross-talk at the level of effector genes

In neural precursor cells, both the STAT and Notch pathways are implicated in the promotion of astrocyte differentiation. Recently a surprising connection was found in which the transcription factor Hes1 has the role of gathering STAT3 with its kinase JAK274 (in a manner similar to the role of growth factor and cytokine receptors; see Figure 17.9, page 525). In short, Notch induces expression of Hes1 in cortical neuroepithelial cells of mouse embryos. Hes1 then binds either STAT3 or JAK2, most likely in the nucleus. Dimerized forms of Hes1 bring STAT3 together with JAK2. The ensuing phosphorylation causes STAT3 dimerization so that they bind DNA and thus act as transcriptional activators (Figure 22.17). This sequence of events shows that growth factors and Notch act in concert to induce STAT3-mediated cellular responses.

Notch and disease

Defects in Notch2 or Jagged1 are the cause of the Alagille syndrome type 2. This is an autosomal dominant multisystem disorder, defined clinically by hepatic bile duct paucity leading to blockage of bile secretion (cholestasis, children having very pale faeces) in association with cardiovascular (stenosis of lung arteries), skeletal (butterfly-shaped vertebral discs), and ophthalmic manifestations. There

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FIG 22.15  Cross-talk between Notch and the Wnt pathway.

In Drosophila, Notch binds Dishevelled and Armadillo, components of the Wnt pathway, and thereby increases the threshold of Wnt signalling. This may constitute a mechanism to filter out weak signals (i.e. reduce noise).

FIG 22.16  Cross-talk between Notch and the TGF signal pathway.

The Nicd fragment of Notch interacts with phosphoSmad1/Smad4 and together they bind CSL and drive expression of Hey2. Hey2 dimerizes and binds the promoter of id1, leading to its suppression. With the onset of cell–cell contact, Delta-mediated Notch signalling arrests BMP4-mediated cell migration.

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