Notch signalling in hematopoiesis

The Notch pathway is a widely utilized, evolutionarily conserved regulatory system that plays a central role in the fate decisions of multipotent precursor cells. Notch often acts by inhibiting differentiation along a particular pathway while permitting or promoting self-renewal or differentiation along alternative pathways. Haematopoietic cells and stromal cells express Notch receptors and their ligands, and Notch signalling affects the survival, proliferation, and fate choices of precursors at various stages of haematopoietic development, including whether haematopoietic stem cells self-renew or differentiate, common lymphoid precursors undergo T or B cell differentiation, or monocytes differentiate into macrophage or dendritic cells. These findings suggest that the Notch pathway plays a fundamental role in regulating haematopoietic development.     

Epidermal Notch signalling: differentiation, cancer and adhesion

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.     

Inhibition of gamma-secretases alters both proliferation and differentiation of mesenchymal stem cells

INTRODUCTION: Human mesenchymal stem cell (hMSC) proliferation and development is regulated by many signalling pathways. gamma-Secretases play an important role in Notch signalling as well as other processes that are involved in developmental decisions, but their role in hMSC proliferation and cell fate decisions has not been explored. OBJECTIVE: To investigate the role of gamma-secretases in hMSC proliferation and differentiation. MATERIALS AND METHODS: Using the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), we investigated their role in hMSC growth and differentiation to chondrogenic, osteogenic and adipogenic fates. RESULTS: We found that inhibiting gamma-secretases reduced the rate of hMSC proliferation, and altered hMSC differentiation in vitro. Addition of DAPT had an inhibitory effect on chondrogenesis resulting in impaired cartilage matrix production and altered chondrocyte morphology. DAPT treated chrodrocytic pellets had reduced levels of Hes1 and Hey1 suggesting that these effects are mediated via Notch signalling. Addition of the DAPT inhibitor to osteogenic cultures did not alter the appearance of bone markers, however, adipogenesis occurred in these cultures in a DAPT concentration-dependent manner. DAPT did not enhance adipogenesis in the presence of a potent adipogenic cocktail, but had an adipogenic effect when combined with dexamethasone only. CONCLUSION: We conclude that gamma-secretases play an important role in both hMSC proliferation and differentiation.     

γ-Secretase-regulated signaling pathways, such as notch signaling, mediate the differentiation of hematopoietic stem cells, development of the immune system, and peripheral immune responses

Notch signaling mediates the fates of numerous cells in both invertebrates and vertebrates. In the immune system, Notch signalling contributes to the generation of hematopoietic stem cells (HSCs), the promotion of HSC self-renewal, T lineage commitment, intrathymic T cell development, and peripheral lymphocyte differentiation/activation. The intracellular domain (ICD) of Notch is released from the cell membrane by γ-secretase and translocates to the nucleus to modulate gene expression. Hence, γ-secretase plays a central role in the regulation of Notch signaling. More than five dozen type 1 transmembrane proteins, including amyloid precursor protein, Notch, and Delta, are substrates for γ-secretase and their ICDs are released from the cell membrane. Therefore, it is highly possible that mechanisms similar to Notch signaling may widely contribute to γ-secretase-regulated signaling. Besides Notch, some transmembrane proteins such as CD44 and CSF-1R, which are important for immune responses, have been reported as substrates for γ-secretase. Since the ICDs of these proteins are also released by γ-secretase from the cell membrane and localize to the nucleus, it is thought that these ICDs modulate gene expression. Thus, γ-secretase-regulated signaling, including Notch signaling, may play a wide range of roles in the immune system.     

Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters

BACKGROUND: Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS: Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS: We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.     

Notch signalling during peripheral T-cell activation and differentiation

For many years, researchers have focused on the contribution of Notch signalling to lymphoid development. Only recently have investigators begun to ask what role, if any, Notch has during the activation and differentiation of naive CD4(+) T cells in the periphery. As interest in this issue grows, it is becoming increasingly clear that the main role of Notch signalling, to regulate cell-fate decisions, might also be influential in peripheral T cells.     

Notch signalling inhibits the adipogenic differentiation of single‐cell‐derived mesenchymal stem cell clones isolated from human adipose tissue

ADSCs (adipose‐derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue‐derived single‐cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose‐derived single‐cell‐clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ‐secretase inhibitor, DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐l ‐alanyl]‐(S)‐phenylglycine t‐butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft‐tissue augmentation application.     

Notch promotes neural lineage entry by pluripotent embryonic stem cells

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.     

Mind bomb-1 in dendritic cells is specifically required for Notch-mediated T helper type 2 differentiation

In dendritic cell (DC)-CD4(+) T cell interaction, Notch signaling has been implicated in the CD4(+) T cell activation, proliferation, and subset differentiation. However, there has been a lot of debate on the exact role of Notch signaling. Here, we observed that expression of Mind bomb-1 (Mib1), a critical regulator of Notch ligands for the activation of Notch signaling, increases gradually as precursor cells differentiate into DCs in mice. To clarify the role of Mib1 in DC-CD4(+) T cell interactions, we generated Mib1-null bone marrow-derived DCs. These cells readily expressed Notch ligands but failed to initiate Notch activation in the adjacent cells. Nevertheless, Mib1-null DCs were able to prime the activation and proliferation of CD4(+) T cells, suggesting that Notch activation in CD4(+) T cells is not required for these processes. Intriguingly, stimulation of CD4(+) T cells with Mib1-null DCs resulted in dramatically diminished Th2 cell populations, while preserving Th1 cell populations, both in vitro and in vivo. Our results demonstrate that Mib1 in DCs is critical for the activation of Notch signaling in CD4(+) T cells, and Notch signaling reinforces Th2 differentiation, but is not required for the activation or proliferation of the CD4(+) T cells.     

Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.     

Expression of Notch receptors and ligands on immature and mature T cells

Notch plays multiple roles in T cell development in the thymus and T cell differentiation in the periphery. In order to systematically examine the role of Notch in T cell biology, we determined the cell surface expression of all Notch receptors and ligands on various populations of T cells by using a panel of specific monoclonal antibodies we recently established. Notch1 and Notch3 were upregulated at double-negative (DN) 2-DN4 stages of immature thymocytes, then downregulated on mature single-positive thymocytes and peripheral T cells, but were rapidly upregulated again upon activation. Notch2 was consistently expressed on T cells while Notch4 was not. Jagged1 and Jagged2 were expressed at double-positive stage of immature T cells. Jagged2 was also inducible on mature T cells upon activation. In contrast, no Delta-like (Dll) 1 or Dll4 expression was observed on T cells. These comprehensive profiling of the expression of Notch receptors and ligands would be informative to fully understand the role of individual Notch receptors and ligands in T cell development and differentiation.     

Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals

Background  

Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways.     

Pharmacological Inhibition of Glycogen Synthase Kinase 3 Regulates T Cell Development In Vitro

The development of functional T cells requires receptor-mediated transition through multiple checkpoints in the thymus. Double negative 3 (DN3) thymocytes are selected for the presence of a rearranged TCR beta chain in a process termed β-selection which requires signalling via the pre-TCR, Notch1 and CXCL12. Signal integration by these receptors converges on core pathways including the Phosphatidylinositol–3-kinase (PI3K) pathway. Glycogen Synthase Kinase 3 (GSK3) is generally thought to be negatively regulated by the PI3K pathway but its role in β-selection has not been characterised. Here we show that developmental progression of DN3 thymocytes is promoted following inhibition of GSK3 by the synthetic compound CHIR99021. CHIR99021 allows differentiation in the absence of pre-TCR-, Notch1- or CXCL12-mediated signalling. It antagonizes IL-7-mediated inhibition of DP thymocyte differentiation and increases IL-7-promoted cell recovery. These data indicate a potentially important role for inactivation of GSK3 during β-selection. They might help to establish an in vitro stromal cell-free culture system of thymocyte development and offer a new platform for screening regulators of proliferation, differentiation and apoptosis.     

Notch signalling in B cells

Notch signalling is likely to regulate multiple aspects of lymphoid development and function. During T cell development, Notch signalling is required for commitment of the earliest progenitor, and may also function during other developmental stages. T cell commitment from a common lymphoid progenitor occurs at the expense of B cell development, suggesting that Notch signalling inhibits the earliest stage of B lymphopoiesis. In contrast, recent evidence suggests that Notch promotes the development of marginal zone lymphocytes. Not only is Notch required for later stages of B cell development, but several viral proteins appear to utilize Notch signalling in B cells to mediate their functions. In this review, we will focus on potential roles of Notch signalling in B lymphopoiesis and also consider how viral proteins may utilize Notch signalling in B cells.